Rapid and sensitive method for detection of biological targets

ABSTRACT

The present invention relates to a method of biological labeling that occurs via a free radical chain reaction. The labeling occurs due to deposition of a detectable reporter molecule from a media comprising a substance comprising at least two moieties of a peroxidase enzyme substrate (termed herein ‘cross-linker’) in a target site comprising peroxidase activity and a biological marker. The labeling reaction described herein may generally be used to detect targets in a host of experimental schemes for detecting and visualizing a biological or chemical target, including immunohistochemistry (IHC), in situ hybridization (ISH), antibody-based staining methods such as ELISA, Southern, Northern, and Western blotting, and others.

PRIORITY CLAIM

This application is a continuation of U.S. application Ser. No.12/678,937, filed Jul. 12, 2010, which is a national stage filing under35 U.S.C. §371 of International Application No. PCT/DK2008/000327, filedSep. 16, 2008, and designating the United States of America, whichclaims benefit of the filing date of and right of priority to U.S.Provisional Application No. 60/994,206, filed Sep. 18, 2007, thecontents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a method of biological labeling thatoccurs via a free radical chain reaction. The labeling reactiondescribed herein may generally be used to detect targets in a host ofexperimental schemes for detecting and visualizing a biological orchemical target, including immunohistochemistry (IHC), in situhybridization (ISH), antibody-based staining methods such as ELISA,Southern, Northern, and Western blotting, and others.

BACKGROUND

Detection of biological or chemical targets in a sample using adetectable label is a procedure at the heart of many biologicaldiagnosis and detection methods. In some cases the target may be aparticular polynucleotide sequence or gene, a mutation of a gene, agenetic expression pattern, detected at the DNA or RNA level, either insitu or after extraction or isolation. In other cases, the target may bea peptide, protein, antigen, or other substance, again detected in situor after isolation or laboratory manipulation. The target may also be aparticle or debris of organic origin.

Many standard detection methods, e.g. IHC, ISH, ELISA or blotting,employ labeling schemes to detect the desired targets. Typically, thoseschemes involve incubating an experimental sample potentially containingthe detectable target with a probe, and then detecting the bindingbetween probe and target with a detectable label which may give off acolor, a fluorescent signal, or radioactivity, for example. One or manyprobe molecules may bind to each target, depending upon the specifics ofthe scheme used. In some cases, especially when the target is present inlow concentration, it is necessary to amplify the signal from thetarget-probe binding by adding one or more amplification layers to thesystem. For example, if the probe is a primary antibody that recognizesthe target, a secondary antibody that recognizes the primary antibodyprobe may be added such that many secondary antibodies bind to eachprimary antibody. If the secondary antibodies are attached to adetectable label such as a fluorophore or chromophore, then, viaamplification, each target molecule in the sample may effectively bebound to multiple fluorophores or chromophores instead of only one or afew fluorophores or chromophores. Hence, the target will produce astronger detection signal after amplification.

Some detection experiments, however, have a tendency to producerelatively diffuse-looking signals, especially if the sample is allowedto rest for a period of time before analysis. For example, the one ormore probes and/or detectable labels bound to a target may slowlydiffuse away from the target, or away from each other over time. In somecases buffer changes that affect the binding affinity of the target,probe, and amplification layers can also cause signal diffusion. Manydetectable labels are bound to targets by non-covalent interactions suchas protein-ligand binding or polynucleotide hybridization. Bufferchanges after labeling may reduce the affinity between the target,probe, and detectable label, causing the various components todissociate. Simple diffusion over a period of time, such as severaldays, may also cause dissociation between target, probe, and detectablelabel, rendering the signal diffuse.

Prior art describes only a very few techniques which allow to overcomethe above mentioned problems, but yet only partially. One example ofsuch techniques is a method of catalyzed reporter deposition (CARD)described in U.S. Pat. Nos. 5,863,748; 5,688,966; 5,767,287; 5,731,158;5,583,001, 5,196,306, 6,372,937 or 6,593,100. This method utilizesso-called “analyte-dependent enzyme activation system” (ADEAS) tocatalyze the deposition of a detectable label onto the solid phase of anassay platform. In the assay format, an enzyme comprised by the ADEASreacts with a conjugate consisting of a detectably labeled substratespecific for the enzyme. When the enzyme and the conjugate react, anactivated conjugate is formed which deposits covalently at a site wherea specific receptor for the activated conjugate is immobilized. Thus,because of the conjugate comprises a label it plays a role of a reporterwhich indicates the presence of a target in the site. Enzymaticallydeposited labels may be detected directly or indirectly. The methodresults in signal amplification and improved detection limits.

The CARD method may be used in assay formats, where the target to bedetected is a receptor immobilized on a solid support, e.g. a membrane.Such assays formats include sandwich immunoassays and membrane basednucleic acid hybridization assays. The CARD method is also applicable todetection of biological targets e.g. by immunohystochemistry (IHC), asdescribed in U.S. Pat. No. 6,593,100. The method described in U.S. Pat.No. 6,593,100 utilizes a reaction of horse radish peroxidase (HRP) witha labeled conjugate comprising a HRP substrate in the presence of anenhancer. Both HRP substrate and enhancer are derivatives of phenol.Upon reaction with HRP the HRP substrate becomes activated and binds toreceptor sites of the sample, e.g. proteins.

Despite of having some advantageous features, e.g. an increasedsensitivity of detection, the method is limited to reporter moleculeswhich are labeled HRP substrates selected either from tyramide orp-hydroxycinnamic acid or derivatives thereof.

The present invention overcomes the limitations of the above describedCARD method and provides a novel method for a rapid and sensitivedetection of biological and chemical markers. The method comprises bothvaluable features of the CARD method and new features that make itapplicable to a wider range of the assay formats and independent from anarrow selection of reporter molecules and allow a rapid, precise andsensitive detection of a variety of biological or chemical targets.

SUMMARY OF INVENTION

The present invention is based on finding that a variety of moleculescan be deposited from a solution that comprises a substance comprisingat least two moieties of a peroxidase enzyme substrate (termed herein“cross-linker”) in a site comprising a peroxidase activity, e.g. in asite comprising a moiety of a peroxidase enzyme, e.g. HRP. Thedepositing molecule may be a detectable molecule, e.g. a molecule thatitself may give off a color, a fluorescent signal, or radioactivity orthat comprises a detectable label which may give off a color, afluorescent signal, or radioactivity, accordingly, the site ofdeposition of this detectable molecule can be detected, and if thedeposition site comprises a biological or chemical marker, the presenceof this biological or chemical marker can be detected as well. Thedepositing molecule will thus “report” the presence of the biologicalmarker in the site of its deposition. Accordingly, such detectabledepositing molecules are termed herein “reporters”.

One possible reason for the reporter molecule is deposited from a mediacomprising the cross-linker in the presence of peroxidase activity isthat a free-radical chain reaction initiated by the reaction between theperoxidase and the cross-linker is taking place in the media. Freeradicals of the cross-linker molecules formed in the course of thisreaction may prime reporter molecules present in the same media; theprimed reporter molecules may further react to each other and form largeinsoluble aggregates which are deposited in or around the sitescomprising peroxidase activity (termed herein “target sites”). Becauseof the peroxidase activity is strictly localized to target sites, theresult of this chain reaction is that the reporter molecules aredeposited only in the target sites or in a very close proximity to thesesites. If such target sites comprise a biological or chemical marker,e.g. a protein or nucleic acid, the marker may thus be detected bydetecting the deposited reporter.

It was surprisingly found that molecules comprising at least twomoieties of a peroxidase enzyme substrate can play a role ofcross-linker in the method of the invention. Accordingly, the term“cross-linker” is used herein to designate a molecule which comprises atleast two moieties of a molecule (or at least two moieties of twodifferent molecules) which can serve as substrate of a peroxidase (theterm “peroxidase” is interchangeably used herein with the term“peroxidase enzyme” or “peroxidase activity”), and which is capable ofcross-linking activity when activated by the peroxidase. Thecross-linkers of the present invention are capable of cross-linking ofat least two reporter molecules.

It was found that deposition of the reporter mediated by the reaction ofa cross-linker and peroxidase is very rapid and site-directed, i.e. thereporter is deposited non-randomly but specifically in a target sitewhich comprises peroxidase activity, e.g. a moiety of HRP. The depositedreporter is tightly attached to the target site and does not diffusefrom the site over time. Accordingly, a signal associated with thedeposited reporter is precisely localized and stays sharp over time.

Accordingly, a first aspect of the invention is a method for the targetsite-directed deposition of a reporter, wherein said method comprisingincubating a target site in a medium comprising a reporter and across-linker, wherein said target site comprises a peroxidase activity,wherein said reporter is a detectable molecule, and wherein saidcross-linker is a molecule comprising at least two moieties of aperoxidase enzyme substrate.

Another aspect of the invention relates to a media for the site-directedreporter deposition, wherein said media is a water buffered solutionhaving pH from about 4 to about 9, comprising

10⁻⁵-10⁻²M a cross-linker comprising at least two moieties of aperoxidase substrate,

0.1-10 mM a peroxide compound,

0-20% an organic modifier, and

0-2 M a salt.

Another aspect of the invention relates to a method of detection of abiological marker in a sample in vitro, wherein said method comprises astep of site-directed deposition of a reporter described herein. Themethod may advantageously be used as in manual as in automatedprocedures for the detection of biological markers. In particular, theinvention The method of detecting a biological marker in a biologicalsample in vitro according to the invention comprises the followingsteps:

a) incubating a sample presumably comprising a biological marker withone or more probes, wherein the least one of said one or more probescomprises at least one moiety of horse radish peroxidase (HRP), therebyforming a complex of the biological marker with the at least one probecomprising at least one moiety of HRP, i.e. forming a target site;

b) incubating the sample comprising the complex of the biological markerwith the at least one probe comprising at least one moiety of HRP ofstep (a), i.e. the sample comprising a target site, in a mediacomprising a reporter and a cross-linker, thereby depositing thereporter in the target site, i.e. the site where the complex of (a) ispresent;

c) detecting the deposited reporter of (b) and thereby detecting thebiological marker

Further aspect of the invention relates to a cross-linker molecule whichcan be advantageously used for the site-directed deposition of areporter, wherein said molecule has the following formula:

(R1)n-(X)q-R2(m), wherein

R1 and R2 are moieties of a peroxidase enzyme substrate, X is a linkergrouping of the following formula

wherein R3 and R4 are residues of amino acid lysine, and m, n and q areintegers from 1 to 10.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows examples of the cross-linker molecules of the invention

FIG. 2 shows examples of the reporter molecules of the invention.

FIG. 3 shows a schematic presentation of the method of detection of abiological marker of the invention applied for the detection of twodifferent markers.

FIG. 3(1) shows incubating a sample with a first probe 1 (1P1) e.g. anHRP-conjugated antibody). FIG. 3(2) shows incubating the sample of FIG.3(1 with reporter 1 (R1) (e.g. a ferulic acid-PNA1 conjugate) in thepresence of a cross-linker (e.g. DAB). FIG. 3(3) shows incubating thesample of FIG. 3(2) with hydrogen peroxide (e.g. at >3% v/v whichquenches the residual HRP activity as shown b the X over the HRP at theAI site in FIG. 3(4). FIG. 3(4) shows incubating the sample of FIG. 3(3)with first probe 2 (1P2) (e.g. HRP-conjugated antibody AB2). The firstpart of FIG. 3(5) shows incubating the sample FIG. 3(4) with reporter 2(R2) (e.g. ferulic acid-PNA2 conjugate) and DAB, resulting in thedeposition of report 2 at the A2 site, as shown in the second part ofFIG. 3(5). The first part of FIG. 3(6) shows incubating the sample shownin the second part of FIG. 3(5) with second probe 1 (2P1) (e.g.PNA1′-FITC) and with second probe 2 (2P2) (e.g. PNA2′-Texas Red), withthe results shown in the second part of FIG. 3(6). That is the secondpart of FIG. 3(6) shows an example of when a green fluorescent signal(emanating from PNA1′-FITC) may be detected from the target site whereR1 is deposited, a red fluorescent signal (emanating from PNA2′-TexasRed) may be detected from the target site of R2 deposition, resulting ina yellow signal where both R1 and R2 are deposited, i.e., from the sitewere both targets AI and A2 are present, as shown in the second part ofFIG. 3(6).

DETAILED DESCRIPTION OF INVENTION 1. Method of Site-Directed Depositionof a Reporter

In one aspect the present invention relates to a method of deposition ofa reporter in a target site, said method comprising incubating a targetsite in a medium comprising a reporter and a cross-linker, wherein saidtarget site comprises a peroxidase activity, wherein said reporter is adetectable molecule, and wherein said cross-linker is a moleculecomprising at least two moieties of a peroxidase enzyme substrate

Cross-Linker

The term “cross-linker” is used herein to refer to a molecule capable oflinking together at least two molecules, e.g. at least two reportermolecules. The cross-linker of the invention comprises at least twomoieties of a peroxidase enzyme substrate, wherein said two moieties maybe bound to each other by a chemical bond, or may be liked via a linkingmolecule or grouping.

Accordingly, the invention relates to a cross-linker which a compound ofthe following formula:

(R1)n-(X)q-R2(m),

wherein

R1 and R2 are moieties of a peroxidase enzyme substrate

X is a linker grouping or a chemical bond, and

m, n and q are integers from 1 to 10.

In one embodiment the cross-linker of the invention may be a compound ofthe following formula:

R1)n-(X)q-R2(m),

wherein R1 and R2 are moieties of a peroxidase enzyme substrate,

X is a chemical bond, and

m, n and q are integers selected from 1 to 10.

One non-limiting example of such cross-linker, e.g. wherein X is acovalent bond and each of m, n, and q is 1, may be 3,3 diaminobenzidine(DAB). DAB comprises two moieties of horse radish peroxidase (HRP)substrate o-phenylenediamine (OPD) (i.e. R1 and R2 are the OPD moieties)which are linked to each other via one covalent bond.

In another embodiment, the invention relates to a cross-linker of theformula

(R1)n-(X)q-(R2)m, wherein R1 and R2 are moieties of a peroxidase enzymesubstrate, X is a linking molecule or a linking grouping, and m, n and qare integers from 1 to 10.

In such cross-linker the (X) grouping may be any linker molecule orlinking grouping. In one preferred embodiment X may be a linkinggrouping of the formula:

wherein R3 and R4 are residues of amino acid lysine. Such linkergrouping is termed herein L30.

In some embodiments the cross-linkers may comprise many moieties of aperoxidase substrate (n and/or m more than 2) and several moieties ofthe linking molecule X (i.e. q>1). In other embodiments linkers maycomprise a fewer moieties of a peroxidase substrate (each of n and niare 1) and several moieties of the linking molecule X (i.e. q>1).

In some embodiments the cross linker may be a compound in which severalmoieties of a peroxidase substrate bound directly to each other viachemical bonds. The moieties R1 and R2 may in one embodiment be moietiesof the same peroxidase enzyme substrate. In another embodiment R1 and R2may be moieties of two or more different peroxidase enzyme substrates.

An example of a cross-linker, wherein the linking grouping (X) isrepresented by a dimer of the L30 linker and R1 and R2 are multiplemoieties of a HRP substrate may be compound D17140 which is demonstratedin FIG. 1 and described in Example 1.8. Another example of thecross-linker of the invention may be compound D17120 described inExample 1.5. The molecule of D17140 comprises 6 moieties of HRPsubstrate ferulic acid attached through lysine residues to a polymermade of several moieties of L30. The molecule of D17120 also comprises 6moieties of ferulic acid attached to another L30 polymer.

By varying the number of peroxidase substrate moieties, linker groupingrepeats and/or introducing charged moieties into cross-linker molecules,e.g. lysine residues such as in the D17140 molecule, it is possible tomake cross-linkers with desirable features, e.g. cross-linkers having agood solubility in the media comprising a reporter.

Thus, in one preferred embodiment the cross-linker may be a moleculewhich comprises at least two moieties of a substrate of a peroxidasewherein bound to a each other via a covalent bond, in another preferredembodiment the cross-linker may be a molecule which comprises more thantwo moieties of a substrate of a peroxidase, wherein said moieties arelinked together via one or more linking groupings.

In particular, in one preferred embodiment the moieties of theperoxidase substrate are moieties of o-phenylenediamine, in anotherpreferred embodiment the moieties are the moieties of ferulic acid. Inthe latter embodiment it is preferred that the moieties of ferulic acidare linked together through one or more molecules of the L30 linker.

Reporter

The term “reporter” is used herein to refer to any detectable molecule,wherein the detectable molecule is a molecule selected from a moleculewhich can give off a color, a fluorescent signal, or radioactivity, orit is a member of a specific binding pair, or it is a conjugatecomprising a detectable moiety, such as chromogenic, fluorescent,chemiluminescent, radioactive label, enzyme moiety, enzyme substrate,detectable particle, etc., or it is a molecule, which can be depositedform the media comprising a cross-linker (embodiments of thecross-linker are described above) in the presence of peroxidase activityand labeled as deposited.

A substance which has the cross-linking capability according to theinvention may in one embodiment be used either as crosslink-linker orreporter; however, one and the same molecule may not be used in the sameembodiment both as cross-linker and reporter.

A cross-linker of the formula (R1)n-(X)q-(R2)m,

wherein R1 and R2 are moieties of o-phenylenediamine,

X is a covalent bond, and

m, n and q are 1, may not be used as reporter in any embodiments of theinvention.

Non-limiting examples of molecules that may be used as a cross-linker inone embodiment and reporter in another embodiment are the molecules017120 and D17140 described above (see also in Examples 1.1 to 1.8.)

The reporter molecule according to the invention is a molecule which issoluble in the media comprising a cross-linker in the absence ofperoxidase activity. Concentration of the reporter in the media may varyfrom about 10⁻⁹ M to about 10⁻⁴M, for example from about 10⁻⁹ M to about10⁻⁸ M, such as from about 10⁻⁸ M to about 10⁻⁷ M, from about 10⁻⁷ M toabout 10⁻⁸ M, or from about 10⁻⁸ M to about 10⁻⁸ M, or from about 10⁻⁸ Mto about 10⁻⁴ M.

The reporter in one embodiment may be a detectable small molecule, e.g.selected from a fluorescent or chromogenic substance, hapten or enzymesubstrate, or in another embodiment the reporter may be a largemolecule, e.g. a conjugate comprising a backbone polymer and at leastone detectable substance, wherein the detectable substance, i.e.detectable label, is attached to the backbone polymer via a chemicalbond or via a linker molecule. Thus, the reporter may be a conjugatecomprising two or more molecules, at least one of which is detectable,or the reporter may be a small molecule, such as a molecule which hasthe molecular mass that is not greater than 500-2000 Da, for exampleabout 1000 Da. The reporter-conjugate may be a large molecule, typicallya polymer molecule to which a detectable label is attached. The size ofsuch reporter molecules may be very different and vary from 3×10³ Da to3×10⁶ Da or more.

The reporter according to the invention may also be a molecule which isnot “detectable”. Such molecule cannot generate a signal which can bedetected, e.g. by means for detection of color, fluorescence orradioactivity. Such reporter molecule can be detected when deposited byapplication of secondary means allowing the detection, e.g. detectablenon-reporter molecules which can specifically bind to the depositedreporter. Such detection may comprise several steps on which one or moredetectable substances will be applied to bind to this deposited “non-detectable” reporter molecule and thus make it visually detectable.

The reporter is a detectable molecule. It may be a small detectablemolecule, or it may be a large detectable molecule. The small detectablemolecule is typically a directly detectable molecule (some embodimentsof small detectable molecules are described in this section and in thefollowing sections below). The large detectable molecule is typicallyrepresented by a large, typically, not directly detectable moleculewhich is made detectable upon coupling this molecule to a small directlydetectable molecule, or another detectable label. A large reportermolecule comprising a label is termed herein as “detectable conjugate”.The detectable conjugate according to the invention may comprise two ormore different molecules, at least one of which is a detectable label.

Both small detectable molecule and label comprised by a big reportermolecule may be selected from is a fluorescent, luminescent,bioluminescent, radioactive or chromogenic substance.

A number of fluorescent, luminescent, bioluminescent, radioactive orchromogenic labels may be used. Many of them are commercially available,for example fluorescent stains Alexa Fluors (Molecular Probes) andDyLight Fluors (Thermo Fisher Scientific). Other non-limited examples oflabels and small reporter molecules may be the molecules of the groupconsisting of 5-(and 6)-carboxyfluorescein, 5- or 6-carboxyfluorescein,6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluoresceinisothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, Cy5, AMCA,PerCP, R-phycoerythrin (RPE) allophycoerythrin (APC), Texas Red,Princeton Red, Green fluorescent protein (GFP) coated CdSenanocrystallites, DNP, digoxiginin, ruthenium derivatives, luminol,isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines,radioactive isotopes of hydrogen, carbon, sulfur, iodide, cobalt,selenium, tritium, or phosphor.

In another embodiment a small detectable molecule or label may be asubstance which is an enzyme substrate. An enzyme substrate may beselected form the group consisting of substrates of horse radishperoxidase (HRP), excluding DAB, alkaline phosphatase (AP),beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase,beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthineoxidase, firefly luciferase, glucose oxidase (GO). In one preferredembodiment the enzyme substrate label may be a HRP substrate, in anotherpreferred embodiment the enzyme label may be an AP substrate.

Examples of useful substrates of HRP include 3-amino-9-ethylcarbazole(AEC), Benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR),Indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphtol(CN), α-naphtol pyronin (a-NP), o-dianisidine (OD),5-bromo-4-chloro-3-indolylphosphate (BCIP), Nitro blue tetrazolium(NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride(INT), tetranitro blue tetrazolium (TNBT),5-bromo-4-chloro-3-indoxyl-beta-D-galactoside/ferro-ferricyanide(BCIG/FF),5-amino-2-[3-[5-amino-1,3-dihydro-3,3-dimethyl-1-(4sulfobutyl)-2Hindol-2-ylidene]-1-propenyl]-3,3dimethyl-1-(4sulfobutyl)-3H-Indolium. Inone preferred embodiment the small molecule may be5-amino-2-[3-[5-amino-1,3-dihydro-3,3-dimethyl-1-(4sulfobutyl)-2H-indol-2-ylidene]-1-propenyl]-3,3dimethyl-1-(4sulfobutyl)-3H-Indolium

Examples of useful substrates of AP includeNaphthol-AS-B1-phosphate/fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/fast red TR (NABP/FR),Naphthol-AS-MX-phosphate/fast red TR (NAMP/FR),Naphthol-AS-B1-phosphate/new fuschin (NABP/NF), bromochloroindolylphosphate/nitroblue tetrazolium (BCIPINBT),5-Bromo-4-chloro-3-indolyl-b-d-galactopyranoside (BCIG).

The label or a small reporter molecule may be an enzyme. Non-limitingexamples of enzyme labels may be alkaline phosphatase (AP),beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase,beta-N-acetylglucosaminidase, fl-glucuronidase, invertase, xanthineoxidase, firefly luciferase, glucose oxidase (GO) In one preferredembodiment the enzyme label is AP.

Yet, the detectable label of a conjugate reporter molecule or smallreporter molecule may be a member of a specific binding pair. Members ofspecific binding pairs suitable for use in practicing the invention maybe of the immune or non-immune type. Immune specific binding pairs areexemplified by antigen/antibody systems-or hapten/anti-hapten systems.

Haptens are small molecules thus permitting multiple copies to beattached to a single polymer molecule, in case the reporter comprisesboth a detectable label and polymer.

Haptens provide convenient target molecules for assay formats where itis necessary or advantageous to amplify a signal. Thus, the boundmultiple copies of a hapten provide for enhanced sensitivity, e.g.increased signal strength. Examples of suitable haptens include FITC,DNP, myc Digoxigenin, nitrotyrosine biotin, avidin, strepavidin andanti-dye antibodies to e.g. tetramethylrhodamine, Texas Red, dansyl,Alexa Fluor 488, BODIPY FL, lucifer yellow and Alexa Fluor 405/CascadeBlue fluorophores.

The antibody member, whether polyclonal, monoclonal or an immunoreactivefragment thereof, of the binding pair can be produced by customarymethods familiar to those skilled in the art. The terms immunoreactiveantibody fragment or immunoreactive fragment mean fragments whichcontain the binding region of the antibody. Such fragments may beFab-type fragments which are defined as fragments devoid of the Fcportion, e.g. Fab, Fab′ and F(ab′), fragments, or may be so-called“half-molecule” fragments obtained by reductive cleavage of thedisulfide bonds connecting the heavy chain components of the intactantibody. If the antigen member of the specific binding pair is notimmunogenic, e.g. a hapten, it can be covalently coupled to a carrierprotein to render it immunogenic.

Non-immune specific binding pairs include systems wherein the twocomponents share a natural affinity for each other but are notantibodies. Exemplary non-immune binding pairs are biotin-avidin orbiotin-streptavidin, folic acid-folate binding protein, complementarynucleic acids, receptor-ligand, etc. The invention also includesnon-immune binding pairs which form a covalent bond with each other.Exemplary covalent binding pairs include sulfhydryl reactive groups suchas maleimides and haloacetyl derivatives and amine reactive groups suchas isothiocyanates, succinimidyl esters, sulfonyl halides, and coupler20 dyes such as 3-methyl-2-benzothiazolinone hydrazone (MBTH) and3-(dimethyl-amino)benzoic acid (DMAB), etc.

In some embodiments, it may be preferred that labels linked to a polymermolecule are different. In some embodiments it may be preferred to use areporter that comprises two or more different labels. Any combination ofdifferent labels selected from any of the groups identified above may bemade, e.g. a reporter may comprise a combination of a fluorescent labeland enzyme label, a combination of a member of a specific binding pair,enzyme and/or enzyme substrate, etc.

If the reporter is represented by a conjugate of a polymer with one ormore detectable molecules, in one embodiment the conjugate may compriseat least one polymer and at least one label, wherein the at least onelabel is linked to the at least one polymer via a chemical bond or via alinker groping, e.g. L30. Non-limited examples of such reporter aredescribed in Examples, see for example Example 1.9. If the conjugatecomprises more than one polymer, each of the polymers may be linked toone or more detectable labels.

The choice of polymer may depend on particular application where themethod of the invention is used. Physical properties of the polymer maybe selected depending on

The labels may be same or different. Different labels may be selectedfrom any groups of the described above and used in any desiredcombinations.

Reporter Molecules Comprising Polymers

A large reporter molecule may comprise a polymer. The polymer comprisedby a large reporter molecule may be any polymeric molecule. It may besoluble or insoluble in water on its own, but when serves as part of areporter conjugate, it is soluble or can be made soluble in a watermedia or at least in the media of the invention described below. Thepolymer is preferably selected from molecules which can be depositedfrom the media comprising a cross-linker in the presence of peroxidaseactivity.

Examples of suitable polymers include polysaccharides such as dextrans,carboxy methyl dextran, dextran polyaldehyde, carboxymethyl dextranlactone, and cyclodextrins; pullulans, schizophyllan, scleroglucan,xanthan, gellan, O-ethylamino guaran, chitins and chitosans such as6-O-carboxymethyl chitin and N-carboxymethyl chitosan; derivatizedcellolosics such as carboxymethyl cellulose, carboxymethyl hydroxyethylcellulose, hydroxyethyl cellulose, 6-amino-6-deoxy cellulose andO-ethylamine cellulose; hydroxylated starch, hydroxypropyl starch,hydroxyethyl starch, carrageenans, alginates, and agarose; syntheticpolysaccharides such as ficoll and carboxymethylated ficoll; vinylpolymers including poly(acrylic acid), poly(acryl amides), poly(acrylicesters), poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(maleic acid), poly(maleic anhydride), poly(acrylamide),poly(ethyl-co-vinyl acetate), poly(methacrylic acid),poly(vinylalcohol), poly(vinyl alcohol-co-vinyl chloroacetate), aminatedpoly(vinyl alcohol), and co block polymers thereof; poly ethylene glycol(PEG) or polypropylene glycol or poly(ethylene oxide-co-propyleneoxides) containing polymer backbones including linear, comb-shaped orhyperbranched polymers and dendrimers, including branchedPAMAM-dendrimers; poly amino acids including polylysines, polyglutamicacid, polyurethanes, poly(ethylene imines), pluriol; proteins includingalbumins, immunoglobulins, and virus-like proteins (VLP), andpolynucleotides, DNA, PNA, LNA, oligonucleotides and oligonucleotidedendrimer constructs. Also contemplated is the use of mixed polymers,i.e., a polymer comprised of one or more of the above examples includingany of the polymers, the co-block polymers and random co-polymers.

The choice of polymer may depend on particular application where themethod of the invention is used. Physical properties of the polymer maybe selected depending on particular applications of the method tooptimize the performance. Examples of these physical properties includethe length and branching of the polymer. Furthermore, the polymer maycarry various substituents. The substituents may be chemically protectedand/or activated, allowing the polymer to be derivatized further. Forexample in one embodiment the polymer may be a nucleic acid, in anotherembodiment it may be a nucleic acid analog, in another embodiment it maybe a polypeptide, in another embodiment it may be a polysaccharide, inother embodiments it may be any variant thereof.

By the term “nucleic acid” is meant a polymer composed of a chain(s)nucleotide monomers. The most common nucleic acids are deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). A nucleotide is a compound thatconsists of a heterocyclic base (nucleobase), a sugar, and one or morephosphate groups. In the most common nucleotides the base is aderivative of purine or pyrimidine, and the sugar is the pentose(five-carbon sugar) deoxyribose or ribose. As said, nucleotides are themonomers of nucleic acids. The nucleobases are the parts of nucleotidethat may be involved in pairing in RNA and DNA molecules. Thenucleobases include cytosine, guanine, adenine, thymine (DNA), uracil(RNA).

By the term “nucleic acid analog” is meant a polymer, which is ahomopolymer composed of nucleotide monomers, wherein nucleobases may benatural, modified and/or synthetic, or which is a heteropolymer composedof monomers of natural, modified and synthetic nucleobases, amino acidsand other types of monomers. “Home-” and “hetero-” in respect of apolymer indicates that the polymer is composed of monomer of differentchemical origin, e.g. a polymer composed of nucleobase monomers only isa homopolymer, a polymer composed of nucleobase and amino acid monomersis heteropolymer. An example of a homopolymer may be a DNA or RNAmolecule, an example of a heteropolymer may be a PNA molecule. Peptidenucleic acid (PNA) is a chemical similar to DNA or RNA. PNA isartificially synthesized compound. DNA and RNA have a deoxyribose andribose sugar backbone, respectively, whereas PNA's backbone is composedof repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Thevarious purine and pyrimidine bases are linked to the backbone bymethylene carbonyl bonds. PNAs are depicted like peptides, with theN-terminus at the first (left) position and the C-terminus at the right.Since the backbone of PNA contains no charged phosphate groups, thebinding between PNA/DNA strands is stronger than between DNA/DNA strandsdue to the lack of electrostatic repulsion. Mixed base PNA molecules aretrue mimics of DNA molecules in terms of base-pair recognition. PNA/PNAbinding is stronger than PNA/DNA binding

The term “protein” is used herein interchangeably with the term“polypeptide” and refers to at least one polymer composed of natural orartificial amino acids.

The polymer comprised by a reporter molecule may also be selected from apolysaccharide, pullulan, schizophyllan, scleroglucan, xanthan, gellan,O-ethylamino guaran, chitin, chitosan, derivatized cellolosic,hydroxylated starch, carrageenan, alginate, agarose, syntheticpolysaccharide, vinyl polymer, poly ethylene glycol (PEG) orpolypropylene glycol or poly(ethylene oxide-co-propylene oxides)containing polymer backbones including linear, comb-shaped or brancheddendrimer, poly amino acid, poly(ethylene imine), or pluriol.

In some embodiments the polymer may be a mixed polymer comprised of twoor more different polymers described above.

In some preferred embodiments the polymer may comprise dextran. Inanother preferred embodiments the polymer may comprise at least onenucleic acid. In another preferred embodiments the polymer may compriseat least one nucleic acid analog. In another preferred embodiment thepolymer may consist of or comprise L30 molecule. In other preferredembodiments the polymer may comprise at least one polypeptide.

As already mentioned, the polymer may be conjugated with a detectablelabel. In some embodiments, the detectable label may be conjugated tothe polymer directly, i.e., covalently attached to the polymer, in otherembodiments the detectable label may be conjugated to the polymerindirectly via a linker. Many such linkers are known in the art.Non-limited examples include polyethylene glycol and poly amides. Alinker molecule in one embodiment may comprise 5-15 atoms, in anotherembodiment it may comprise 15-30 atoms, in another embodiment it maycomprise more than 35 atoms, for example 36-45, in some embodiments thelinker may comprise more than 45 atoms. One preferred embodiment of suchlinker molecule is L30 described above.

As mentioned above, L30 in some embodiments may serve as a backbonepolymer of the reporter conjugate molecule. Some of such embodiments aredescribed below in the provided examples (Examples 1.1-1.8) and shown inFIG. 2. In some other embodiments L30 may serve as linker grouping forthe linking detectable labels to the polymer.

In some embodiments, 1-500 detectable label molecules may be directly orindirectly linked to a polymer molecule. In some embodiments, thedetectable label is an enzyme and the number of enzyme molecules linkedto each polymer molecule is 1-200, 2-50, 2-25. In some embodiments, thedetectable label is a gold particle, a dye, a low molecular weightfluorochrome and the number of detectable substances linked to eachpolymer molecule is 1-500, 1-200, 1-100, 10-100, 20-50, 50-100, 1-50,2-30, 10-20. In some embodiments, the detectable label is a proteinfluorochrome, and the number of detectable molecules linked to a polymermolecule is 1-50, 2-20. In some other embodiments the detectable labelis a nucleic acid or nucleic acid analog, e.g., an oligonucteotide orPNA molecule, and the number of detectable molecules linked to a polymeris 1-200, 2-50, 2-25.

Many methods of forming polymeric conjugates are known in the art andcan be used to make the polymeric conjugates of the invention. In someembodiments a detectable substance, if desired, can be chemicallylinked, or conjugated, to a polymeric backbone. In some embodiments, thepolymer conjugate is formed by covalent coupling amino groups toconjugated double bonds. The polymer may be activated with vinylsulfonand mixed with a detectable substance to form the polymer conjugate. Inother embodiments, aldehydes are used to activate a polymeric backbone,e.g., dextrans which are then mixed with a detectable substance. Yetanother method of preparing polymeric conjugates is by using so calledchemo selective coupling schemes for coupling the components together,e.g., enzymes or other molecules can be derivatized with thiol reactivemaleimide groups before being covalent coupled to an thiol modifiedpolymeric carrier or backbone. Other embodiments described below permitthe reagents themselves to form conjugates, e.g. the detectablesubstance.

As mentioned above, the polymer may serve on its own as a reportermolecule and not comprise any detectable label. Non-limited examples ofsuch polymers may be nucleic acids, nucleic acid analogs and proteins.

Small Reporter Molecules

As already discussed above, the reporter may be a small molecule. By“small molecule is meant non-polymeric molecule of not more than 3000Da, typically, from around 200 to around 1000 Da, for example around 500Da. Typically such small reporter molecule is soluble in the media ofthe invention which comprises a cross-linker. The invention relates toany kind of such small molecule which can be deposited from the media inthe presence of a peroxidase.

The small molecule may by a directly detectable substance. Examples ofsuch substances include but not limited to 5-(and 6)-carboxyfluorescein,5- or 6-carboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidohexanoic acid, fluorescein isothiocyanate, rhodamine,tetramethylrhodarnine, Cy2, Cy3, Cy5, AMCA, PerCP, R-phycoerythrin (RPE)allophycoerythrin (APC), Texas Red, Princeton Red, Green fluorescentprotein (GFP) coated CdSe nanocrystallites, DNP, digoxiginin, luminol,isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines, andradioactive isotopes of hydrogen, carbon, sulfur, iodide, cobalt,selenium, tritium, and phosphor. The chromogenic substance may beselected form 3,3′,5,5′-Tetramethylbenzidine (TMB),10-acetyl-3,7-dihydroxyphenoxazine (ADHP), 3-amino-9-ethylcarbazole,4-chloro-1-naphtol (AEC), ophenilenediamine (OPD),2,2″-azido-bis(3)-ethylbenzthiazoline-6-sulphonic acid ,5-amino-243-[5-amino-1, 3-dihydro-3,3-dimethyl-1-(4sulfobutyl)-2H-indol-2-ylidene]-1-propenyl]-3,3dimethyl-1-(4sulfobutyl)-3H-Indolium.In one embodiment, the detectable small molecule may be a substrate fora peroxidase, preferably horse radish peroxidase (HRP). In one preferredembodiment the detectable small molecule is5-amino-2-[3-[5-amino-1,3-3-dimethyl-1-(4sulfobutyl)-2H-indol-2-ylidene]-1-propenyl]-3,3dimethyl-1-25(4sulfobutyl)-3H-Indolium.

In some embodiments the small molecule may be a molecule which cannot bedetected directly, e.g. a substance which is not capable to becomecolored, fluorescent or chemiluminescent, e.g. biotin, or a substrate ofa peroxidase enzyme such as ferulic acid or tyrosine. In suchembodiments a detectable substance may be coupled to this kind of smallmolecule. For example the small molecule and detectable substance may bederivatized, e.g., with vinyl groups. Polymerization occurs by additionof a radical, which results in polymerization of the vinyl groups toform a polymeric conjugate. The conjugate thus will contain a poly vinylbackbone or blocks of poly vinyl. Active esters of acrylic acid can beused to activate the molecules. Generating free radicals can polymerizethe derivatized molecules. Small molecule linkers with more than onevinyl group can be further added to help form a polymeric conjugate of asmall molecule and a detectable substance. In some other embodiments,the small molecule and detectable substance can be derivatized with across binder. Examples of this method include the use ofhomobifunctional cross binders such as glutaric dialdehyde, hexandiisocyanate, dimethylapimidate, 1,5-difluoro-2,4-dinitrobenzene,heterobifunctional cross binders like e.g. N-gamma-maleimidobytyroloxysuccinimide ester, and zero length cross binders such as1-ethyl-3-(3-dimethylaminopropyl)cabodiimide. By choosing the correctreaction conditions, the cross binders can form bridges between variousfunctional groups in, e.g., the detectable substances and detectableagents to form a polymeric reporter molecule.

Peroxidase

Any enzyme which is capable of peroxidase activity is suitable forpracticing the present invention.

According to the invention peroxidase activity is present in a targetsite. The term ‘target site” refers to a site where the reportermolecule is to be deposited, e.g. a site comprising a biological orchemical marker. According to the invention the peroxidase activity isassociated with at least one moiety of a peroxidase enzyme present inthe target site. The term “one moiety” means that the peroxidase may bea natural or recombinant protein or a derivative thereof, e.g. afragment thereof capable of peroxidase activity. In particular, theperoxidase may be selected from horse radish peroxidase (HRP) or soybeanperoxidase (SP), fragments, recombinant or fusion proteins thereof. Inone preferred embodiment the peroxidase is HRP.

In one embodiment the target site may be a site of any solid supportwhich comprises peroxidase activity. Suitable supports include syntheticpolymer supports, such as polystyrene, polypropylene, substitutedpolystyrene, e.g., aminated or carboxylated polystyrene;polyacrylamides; polyamides; polyvinylchloride, etc.; glass beads;agarose; nitrocellulose; nylon; polyvinylidenedifluoride;surface-modified nylon, etc. A peroxidase molecule can be directly orindirectly immobilized on these supports The target site may be a siteof a biological sample, e.g. a site of a cell membrane, cell organelle,in case the biological sample comprises a cell, it may also be a site ofa cell free biological sample, e.g. plasma sample or cell lysate orextract which is immobilized on a solid support such as described above.Such site will typically comprise a biological marker which may be atarget molecule or structure. The peroxidase activity, will typically beassociated with the target molecule indirectly, e.g. as part of aspecific probe bound to the target molecule.

Media

The media of the invention is a media from which a soluble reportermolecule may be deposited in the presence of peroxidase activity. It isa water buffered solution with pH from about 4 to about 9, essentiallycomprising (i) a compound capable of cross-linking of at least tworeporter molecules in the presence of peroxidase activity, wherein saidcompound is a molecule comprising at least two moieties of a peroxidaseenzyme substrate, and wherein at least one of said two reportermolecules is a detectable molecule, and (ii) a peroxide compound.

Suitable soluble reporter molecules comprised in the media is in detaildiscussed above.

Suitable compounds capable of cross-linking of at least two reportermolecules in the presence of peroxidase activity is according to theinvention are also in detail discussed above. In one preferredembodiment the cross-linking compound is DAB.

The amount of the cross-linking compound in the media may vary fromabout 10⁻⁵ to about 10⁻²M, such as from about 10⁻⁵ to about 10⁻³M, orfrom about 10⁻⁴ to about 10⁻²M, or from about 10⁻⁵ to about 10⁻⁴M, orfrom about 10⁴ to about 10⁻³M, Concentration of the cross-linker may beoptimized for different embodiments, e.g. when deposition of differentreporters is concerned.

The media according to the invention comprises a peroxide compound. Theperoxide compound may be selected from organic peroxides such astent-butyl peroxide, ditert-butyl peroxide, peracetic acid, or it may bean adduct of hydrogen peroxide, such as hydrogen peroxide urea adduct.In some embodiments hydrogen peroxide (H₂O₂) may be preferred peroxide.The amount of the peroxide compound in the media is vary from about 10⁻⁴to about 10⁻²M in different embodiments.

The media may further comprise an organic modifier and an organicmodifier and organic or inorganic salt.

The inorganic salt may be selected form e.g. sodium chloride, magnesiumchloride, potassium chloride, calcium chloride, sodium phosphate, orammonium sulfate.

In other embodiments the media may comprise an organic salt, such assodium acetate, ammonium acetate or imidazole salts, e.g. imidazolehydrochloride.

The concentration of salt in the media may range from approximately10⁻³M to saturation, e.g. from approximately 20 mM to approximately 200mM, or from approximately 50 mM to approximately 500 mM. In onepreferred embodiment, the media may comprise salt in the amount fromapproximately 10 mM to 500 mM. In another preferred embodiment themedium may be free of salt.

Typically the pH value of the media may vary from around 4 to around 9.Any buffer with a suitable buffer capacity may be used, e.g. phosphatebuffered saline (PBS), imidazole buffer. Other suitable buffers may befound in Good, NE., et al (1966) Hydrogen ion buffers for biologicalresearch. Biochem. 5(2), 467-477. The pH value of the media may beessential for depositing the reporter; it may be optimized depending onthe nature of the reporter.

The media may in different embodiments further comprise:

-   -   (i) an organic modifier and/or    -   (ii) an enzyme enhancer, and/or    -   (iii) an iron chelator, and/or    -   (iv) a detergent, and/or    -   (v) an anti-microbial agent

By the term “organic modifier” is meant any non water solvent whichenhances the solubility of the reporter. In such embodiments it issufficient that the modifier is present in the media in the amount ofaround 1% (v/v or w/v), however, in some embodiments higherconcentrations of the organic modifier may be required. The organicmodifier may for example be polyethylene glycol (PEG). Other examplesinclude but not limited to organic modifiers selected from the groupessentially consisting of lower alcohols, N-Methyl pyrolidone (NMP),dimethylsulphoxide (DMSO), mono- and diethylene glycol, sulpholane,N,N-dimethylformamide (DMF). In some embodiments it may be advantageousto use polyethylene glycol (PEG), e.g. PEG2000. The concentration ofpolyethylene glycol in the media in these cases may vary from about 0.1%(v/v) to about 20% (v/v), for example from about 1%(v/v) to about 15%,such as 5-10% (v/v).

By the term “enzyme enhancer” is meant any compound which enhances thecatalytic activity of peroxidase. Such enzyme enhancer may be selectedfrom the group essentially consisting of phenylboronic acid derivativesand divalent metal ions such as nickel or calcium. Concentration of theenzyme enhancer may vary from about 10⁻⁷ to about 0⁻³ M.

The iron chelator may be ethylene diamine tetra acetic acid (EDTA) orethylene diamine hydroxyl phenylacetic acid type chelator (EDHPA).Concentration of the iron chelator may vary from about 10⁻⁶ to about10⁻² M.

The detergent may be selected from polyethylenglycol-p-isooctyphenylether (NP-40), a surfactant selected from the surfactants based onpolyoxyethylene sorbitanmonolaurate (Tween), or a surfactant based onblock copolymers (pluronic etc.). Concentration of the detergent mayvary from about 0.001% to about 5%.

According to the invention the composition of the media is a stablesolution. The term “stable” in the present context means that thecapability of the media to serve as a reaction media for theperoxidase-mediated reporter deposition remains to be essentiallyunchanged during substantial periods of time; such as the media mayretain its reactive capability unaffected for at least 4 hours at roomtemperature.

The media can also be preserved for longer periods of time. To prolongthe shelf-life of the media it may be recommended to store the media attemperatures below 20° C., e.g. at 410° C., and/or to add to the mediaan anti-microbial compound. The anti-microbial compound may be anyanti-microbial compound commonly used for such purpose, e.g. sodiumazid, Proclin™ or Bronidox®.

The media described above is a reaction media for depositing a reporterin a target site comprising peroxidase activity, e.g. a site comprisinga biological marker.

2. Method of Detection of a Target in a Biological Sample

The method of deposition of a reporter in a target site described abovemay advantageously be used for detecting biological markers associatedwith this target site, e.g. biological molecules such as nucleic acids,proteins, etc. because the target site may be a site of a biologicalsample, e.g. a site of a cell membrane, cell organelle, in case thebiological sample comprises a cell, or it may be a site of a cell freebiological sample loaded on a solid support, e.g. plasma sample or celllysate or cell extract which are immobilized on a solid support such asdescribed above. The term “biological marker” means in the presentcontext a molecule, molecular complex or structure that is specific fora biological species, cell type, cell compartment, physiologicalcondition, etc. Non-limited examples of such biological marker includebut not-limited to a particular genetic sequence, protein or anotherbiological molecule, chromosomal or membrane structure, virus etc. whichis associated with a particular disease. Biological markers are commonlyused in medical diagnostic as markers of particular diseases andtherapeutic targets.

Accordingly, a further aspect of the invention relates to a method ofdetection of a biological marker in vitro comprising the followingsteps:

-   -   a) incubating a biological sample presumably comprising a        biological marker with one or more probes, wherein the least one        of said one or more probes comprises at least one moiety of        horse radish peroxidase (HRP), thereby forming a complex of the        biological marker with the at least one probe comprising at        least one moiety of HRP, and thereby forming a target site;    -   b) incubating the sample comprising the target site of (a) in a        media comprising a reporter and a cross-linker, thereby        depositing the reporter in the target site, i.e, in the site        where the complex of the biological marker with the at least one        probe comprising at least one moiety of HRP, is present;    -   c) detecting the deposited reporter of (b), and thereby        detecting the biological marker.

Step (a)

Biological sample comprising a biological marker may be any biologicalsample comprising intact or damaged cells, e.g. a body tissue sample orcell lysate.

Non-limiting examples of the biological sample of the invention include:

-   -   a sample of liquid media comprising suspended cells, e.g. blood        sample, clonal cells suspension or suspension of dissociated        cells of a body tissue.;    -   a sample of a body tissue, e.g. a biopsy sample; The tissue        sample may be a fresh tissue sample, or it may be a sample of        preserved tissue, e.g. formalin fixed and paraffin embedded        tissue sample.    -   a sample of a tumor;    -   a sample derived from any living organism, e.g., animal, plant,        bacteria etc. It may comprise eukaryotic cells or prokaryotic        cells or both. It may be a cell smear.    -   a sample comprising viral particles, debris thereof, or viral        products, e.g., viral] 0 nucleic acids, proteins, peptides, etc.

The biological sample presumably comprising a biological marker isaccording to the method of the invention incubated with at least oneprobe comprising at least one moiety of HRP or moiety of anotherperoxidase enzyme.

The term “incubating” means that the sample is maintained in a mediacomprising a probe (or a media comprising a cross-linker and a reporter(step (b)) during a certain period of time. This period of time may varyfrom 1-3 min to 1-2 hours or longer, e.g. overnight. Incubation may beperformed at different temperatures depending on different embodiments,e.g. the type of biological marker molecule to be detected or type ofprobe and/or reporter used for the detection

One probe or more probes which the sample being incubated specificallyrecognize and bind to a biological marker present in the sample.

The probes which recognize the biological marker are capable of specificbinding to this marker and only to this marker. Typically such probesare members of specific binding pairs.

A number of different specific binding pairs are known in the art. Inone embodiment the members of a specific binding pair may be twoantibody molecules. In another embodiment, members of a specific bindingpair may be two complementary nucleic acids. In another embodiment, themembers of a specific binding pair may two nucleic acid analogmolecules. In another embodiment the member of a specific binding pairmay be a members of particular receptor-ligand binding pairs.

A probe which is capable of specifically binding to the biologicalmarker and is the member of a specific binding pair, e.g. a primaryantibody probe, is designated herein as a first probe. The first probemay be optionally labeled with a moiety of peroxidase enzyme.

In one embodiment the first probe may comprise at least one moiety ofHRP or another peroxidase enzyme, e.g. soybean peroxidase (SP). Anon-limiting example of such probe may be a HRP-labeled primary antibodymolecule or a derivative thereof, or HRP-labeled nucleic acid probe.Such first probes specifically bind to the corresponding biologicalmarkers and label these biological markers with peroxidase activity andform thereby target sites.

In another embodiment the first probe may be not labeled, i.e. notcomprise a moiety of a peroxidase. In this embodiment, a moiety ofperoxidase may be linked to a target site through a second probe. Thesecond probe is a probe which is capable of specific binding to thefirst probe, e.g. is the other member of a specific binding pair.Non-limiting examples of such probes may be secondary antibodiesmolecules of derivatives thereof, nucleic acid probes or members ofreceptor-ligand binding pairs. Such second probe may comprise at leastone moiety of HRP or another peroxidase enzyme e.g. soybean peroxidase(SP). By binding to the first probe the second probe comprisingperoxidase activity will label the site where the biological marker isfound with this peroxidase activity and form thereby a target site.

In another embodiment, both the first and second probe may comprise atleast one moiety of peroxidase enzyme, e.g. HRP and/or SP.

The step (a) may in some embodiments include several sub-steps wherethird and fourth probes are used. For example, before proceeding to step(b) of the procedure, the sample comprising a biological marker may besequentially incubated with multiple probes, whereof first probes arecapable of binding to the biological marker, whereas second, third andthe other probes are capable of binding to each other, i.e. secondprobes to first probes, third probes to second probes, etc., and thusone marker molecule will be associated with many different probes. Everyprobe may comprise one or more moieties of a peroxidase enzyme. Suchmulti-probe labeling of a single biological marker may be used when ahigh accumulation of peroxidase activity in a single target site isdesirable. This may be useful for enhancement of reporter deposition ina single target site.

Before proceeding to step (b) of the method, step (a) may be repeated asmany times as desirable in order to increase accumulation of peroxidaseactivity in the target site.

Step (b):

The sample comprising a target site formed on step (a) is furtherincubated in a media comprising a cross-linker and a reporter accordingto the invention.

Details of the composition of the media suitable for the incubation ofstep (b) are discussed above. The composition of the media may varydepending on the species of reporter and cross-linker molecules used andnature of a particular biological marker to be detected. For example,the media may comprise DAB as a cross-linker when the reporter is areporter comprising a polymer backbone to which several fluorescentlabels are attached (e.g. reporter molecules described in Example 6 or7).

Incubation of step (b) according to the invention results in that thereporter molecules present in the incubation media are deposited in thetarget sites, i.e. the sites of a biological sample which comprise abiological marker labeled with peroxidase activity. Because of thereporter is deposited only in or around the site of the presence ofperoxidase activity, the site of its deposition is the site where thebiological marker is present.

In one embodiment, step (b) may comprise at least two incubations:incubation of the sample in the media comprising a cross-linker (i.e.without a reporter); following by (ii) incubation of the sample in themedia comprising both cross-linker and reporter.

Step (b) may optionally be repeated.

Step (c):

As discussed above, the reporter molecule is a detectable molecule.Different embodiments of detectable reporter molecules are describedabove.

Detection of the reporter may be “direct”—the one-step detection in casethe deposited reporter may give off a chromogenic, radioactive orfluorescent signal which can be detected by a suitable means.

Detection may be “indirect”—comprise several steps of detection, e.g.when the deposited reporter is a non-labeled member of a specificbinding pair, e.g. antibody or nucleic acid probe. Such reporter may bedetected by a procedure comprising several steps of detection on whichsteps a number of different detectable probes may be used. Every probeused on any step may comprise multiple detectable labels. The labels mayalso be enzyme labels, e.g. moieties HRP. Such indirect detection of thedeposited reporter in some embodiments may be preferred, e,g. when asignal associated with the deposited reporter in a target site isdesirable to amplify.

A sample comprising the deposited reporter to which a reporterrecognizing probe is bound may be incubated further in the mediacomprising a cross-linker and another reporter. In case the reporterbound probe comprises a moiety of a peroxidase, this further incubationmay be used for deposition of another reporter in the same target site;this may be used for amplification of the initial signal emanating fromthe target site and thus for enhancement of sensitivity of thedetection, or it may be used for labeling the target site with adetectable label which is different from the label used in the initialdeposition. Such further incubation may be repeated several times.

The method of detection of biological targets described above may beused in a variety of assay formats. Some embodiments of these assayformats are described below and illustrated by non-limiting workingexamples of the invention.

Assay formats

Target molecules comprised by cells of a cell suspension may be detectedemploying the method described above in any suitable assay format, forexample in flow cytometry (FC), or ELISA, or immunohytochemistry (IHC),or in situ hybridization (131-1).

In one embodiment the biolodical sample may be a suspension of cells,Target molecules or structures of cells in suspension may be detectedusing FC, ELISA, IHC or ISH. When ELISA, IHC or ISH are used for thedetection cells of the suspension are to be attached to a solid support,e.g. ELISA plate or ICH slide.

In another embodiment the biological sample may be a slice of a bodytissue. Target molecules or structures of cells of such samples will betypically detected using IHC or ISH.

IHC and ISH assay formats usually require a series of treatment stepsconducted on a tissue section mounted on a suitable solid support formicroscopic inspection, or the production of photomicrographs, e.g., aglass slide or other planar support, to highlight by selective stainingcertain morphological indicators of disease states or detection ofbiological markers. Thus, for example in IHC, a sample is taken from anindividual, fixed and exposed to antibodies which specifically bind tothe biological marker of interest. The sample processing steps mayinclude, for example, antigen retrieval, exposure to a primary antibody,washing, exposure to a secondary antibody (optionally coupled to a HRPmoiety), washing, and exposure to a tertiary antibody linked to one ormore HRP moieties. Washing steps may be performed with any suitablebuffer or solvent, e.g., phosphate-buffered saline (PBS), tris bufferedsaline (TBS), distilled water. The wash buffer may optionally contain adetergent, e.g., Tween 20.

As mentioned above, there are in general two categories of histologicalsamples: (1) preparations comprising fresh tissues and/or cells, whichgenerally are not fixed with aldehyde-based fixatives, and (2) fixed andembedded tissue specimens, often archived material.

Before performing detection of a target in the IHC assay format, apre-detection procedure is to be performed. It may involve the steps of:cutting and trimming tissue, fixation, dehydration, paraffininfiltration, cutting in thin sections, mounting onto glass slides,baking, deparaffination, rehydration, antigen retrieval, blocking steps,applying primary antibody, washing, applying secondary antibody enzymeconjugate and washing,

In ISH, a sample is taken from an individual, fixed and exposed to anucleic acid probe which hybridizes by virtue of complementary basepairing to the nucleic acid of interest. The biological sample typicallycomprises a detectable nucleic acid, such as DNA and

RNA, including messenger RNA. Detection of DNA/RNA levels may indicatethe level of expression of a particular gene, and hence may be used todetect a condition (such as a disease condition) of a cell, tissue,organ or organism. The nucleic acid in the sample is typically denaturedto expose binding sites. The probe is typically a double or singlestranded nucleic acid, such as a DNA or RNA, or a nucleic acid analog,such as PNA. The amount of the relevant target protein or nucleic aciddetected by such techniques is then assessed to determine whether it isabove a certain pre-determined minimum threshold or compared to a knownstandard, and therefore, diagnostically relevant. Suitable treatment maythen be planned for the individual if necessary.

Many methods of fixing and embedding tissue specimens are known, forexample, alcohol fixation and formalin-fixation and subsequent paraffinembedding (FFPE).

Fixatives are needed to preserve cells and tissues in a reproducible andlife-like manner. To achieve this, tissue blocks, sections, or smearsare immersed in a fixative fluid, or in the case of smears, are dried.Fixatives stabilize cells and tissues thereby protecting them from therigors of processing and staining techniques.

Any suitable fixing agent may be used, for example, ethanol, aceticacid, picric acid, 2-propanol, 3,3′-diaminobenzidine tetrahydrochloridedihydrate, acetoin (mixture of monomer) and dimer, acrolein,crotonaldehyde (cis+trans), formaldehyde, glutaraldehyde, glyoxal,potassium dichromate, potassium permanganate, osmium tetroxide,paraformaldehyde, mercuric chloride, tolylene-2,4-diisocyanate,trichloroacetic acid, tungstic acid. Other examples include formalin(aqueous formaldehyde) and neutral buffered formalin (NBF),glutaraldehyde, acrolein, carbodiimide, imidates, benzoequinone, osmicacid and osmium tetraoxide.

Fresh biopsy specimens, cytological preparations (including touchpreparations and blood smears), frozen sections and tissues forimmunohistochemical analysis are commonly fixed in organic solvents,including ethanol, acetic acid, methanol and/or acetone.

To facilitate the specific recognition in fixed tissue, it is oftennecessary to retrieve or unmask the targets, i.e., the biologicalmarkers of interest, through pre-treatment of the specimens to increasereactivity of the majority of targets. This procedure is referred to as“antigen retrieval”, “target retrieval” or “epitope retrieval”, “targetunmasking” or “antigen reagents unmasking.” An extensive review ofantigen retrieval (antigen unmasking) may be found in Shi et al. 1997, JHistochem Cytochem, 45(3):327.

Antigen retrieval includes a variety of methods by which theavailability of the target for interaction with a specific detectionreagent is maximized. The most common techniques are enzymatic digestionwith a proteolytic enzyme (for example proteinase, pronase, pepsin,pepsin, trypsin or neuraminidase) in an appropriate buffer or heatinduced epitope retrieval (HIER) using microwave irradiation, heating ina water bath, a steamer, a regular oven, an autoclave or a pressurecooker in an appropriately pH stabilized buffer, usually containingEDTA, EGTA, Tris-HCl, citrate, urea, glycin-HCl or boric acid.Detergents may be added to the HIER buffer to increase the epitoperetrieval or added to the dilution media and/or rinsing buffers to lowernon-specific binding.

The antigen retrieval buffer is most often aqueous, but may also containother solvents, including solvents with a boiling point above that ofwater. This allows for treatment of the tissue at more than 100⁰C atnormal pressure.

Additionally, the signal-to-noise ratio may be increased by differentphysical methods, including application of vacuum and ultrasound, orfreezing and thawing of the sections before or during incubation of thereagents.

Endogenous biotin binding sites or endogenous enzyme activity (forexample phosphatase, catalase or peroxidase) may be removed as a step inthe detection procedure, e.g., endogenous biotin and peroxidase activitymay be removed by treatment with peroxides. Endogenous phosphataseactivity may be removed by treatment with levamisole. Endogenousphosphatases and esterases may be destroyed by heating.

Blocking of non-specific binding sites with inert proteins like, horseserum albumin (HSA), casein, bovine serum albumin (BSA), and ovaibumin,fetal calf serum or other sera, or detergents like Tween20, TritonX-100, Saponin, Brij or Pluronics may be used. Blocking non-specificbinding sites in the tissue or cells with unlabeled and targetnon-specific versions of the specific reagents may also be used.

Samples may also be prepared and target molecules detected using thefree floating technique. In this method a tissue section is brought intocontact with different reagents and wash buffers in suspension or freelyfloating in appropriate containers, for example micro centrifuge tubes.

The tissue sections may be transferred from tube to tube with differentreagents and buffers during the staining procedure using for example a“fishing hook like” device, a spatula or a glass ring. The differentreagents and buffer can also be changed by gentle decantation or vacuumsuction. Alternatively, containers with the tissue sections can beemptied into a special staining net, like the Corning “Netwells”(Corning,) and the tissue section washed before being transferred backinto the tube for the next staining step.

All the steps, including for example fixation, antigen retrieval,washing, incubation with blocking reagents, immuno-specific reagents andthe peroxidase-mediated reporter deposition, are done while the tissuesection is floating freely or withheld on nets. After deposition of thereporter, the tissue section is mounted on slides, the reporter isdetected and slide covered with a cover slip before being analyzed,e.g., by light or fluorescent microscopy.

In some embodiments, the tissue section may be mounted on slidesfollowing the critical incubation with the immuno-specific reagentsfollowing the procedure (a) of the method.

The rest of the process of detection is then conducted on the slidemounted tissue sections.

Detectable Biological Markers

Detectable by the method, the biological marker may be any molecule orstructure present in a sample, preferably in a biological sample, e.g. aprotein, glycoprotein, lipoprotein, phosphoprotein, methylated protein,or a protein fragment, e.g., a peptide or a nucleic acid, e.g., DNA,RNA, it a lipid, a glycolipid, or a sugar, a polysaccharide, or astarch. The biological marker may be expressed on the surface of thebiological sample, e.g., membrane bound. The marker may be contained inthe interior of the biological sample, i.e., within the cell membrane,e.g., within the cytoplasm, within the nucleus, within an intracellularcompartment or organelle. The biological marker may be a cellularstructure, such as a membrane microdomain, ion chanel, chromosomalstructure, etc., or it may be a molecular complex, e.g. RNA-proteincomplex, etc. A biological marker is preferably a specific biologicalmarker, for example it is a marker of a normal or pathologicalcondition, or it is specific for a particular cell or tissue, orspecific for a particular biological species.

Detection of such biological marker may be useful in diagnosis andtreatment of pathological conditions.

The invention relates to detection at least one biological marker in abiological sample. Accordingly the invention also provides for detectingmultiple biological markers. e.g., two, three, in a given sample andthus provides a method of obtaining data, e.g., diagnostic information,concerning expression of biological markers, panels of proteins, genesor combinations of one or more proteins and one or more genes. As anexample, but not as a limitation, HER2 protein and the HER2 gene can bescreened simultaneously in a cancer diagnostic assay, e.g., an assay forbreast cancer. Another non-limiting example may include screening forthree markers, e.g., to detect cervical cancer. The markers may includeKi67/mib-1, as well as the cellular proliferation marker, p16(INK4a),along with a marker, e.g., a protein or nucleic acid, for humanpapilloma virus. Yet another non-limiting example includes screening formultiple markers associated with prostate cancer. These markers mayinclude AMACR P504S, high molecular weight cytokeratin (HMW-CK), andp63. Screening this combination of markers provides a method todistinguish benign prostate tumors from malignant ones.

It is desirable to minimize cross-reactivity between binding agents,e.g. where multiple markers are detected. This can be accomplished byusing different probes and different reporter molecules in the detectionprocedures. An example of such a system is depicted in FIG. 3 where twodifferent biological markers, e.g. two different cellular receptors, aredetected using the following steps:

(1). incubating the sample with first probe 1 (1P1) (e.g. anHRP-conjugated antibody AB1)

(2). incubating the sample (1) with reporter 1 (R1) (e.g. ferulicacid-PNA1 conjugate) in the presence of a cross-linker (e.g. DAB)

(3). incubating the sample (2) with hydrogen peroxide (>3% v/v)

(4). incubating the sample (3) with first probe 2 (1P2) (e.g.HRP-conjugated antibody AB2)

(5). incubating the sample (4) with reporter 2 (R2) (e.g. ferulicacid-PNA2 conjugate

(6) incubating the sample (5) with second probe 1 (2P1) (e.g.PNA1′-FITC) and with second probe 2 (2P2) (e.g. PNA2′-Texas Red)

As the result, the green fluorescent signal (emanating from PNA1′-FITC)is detected from the target site where R1 is deposited, the redfluorescent signal (emanating from PNA2′-Texas Red) is detected from thetarget site of R2 deposition, and the yellow signal where both R1 and R2are deposited, i.e. from the site were both targets Al and A2 arepresent.

All steps of the method (from (a) to (c)) may be completed within 2 -20min. Such rapid detection may be advantageously used for automated orsemi-automated detection of target biological markers. Automatedstaining devices are known in the field and the method may be adaptedfor these devices.

Automated staining devices may be used in various embodiments of theinvention, for example for the detection of multiple biological markers.Detection of multiple markers frequently requires balancing of thesignals emanating from the different detectable substances. Automatedprocedure may include multiple steps of amplification of the signalsemanating from target biological markers. It is especially advantageouswhen multiple markers are to be detected.

Antibody Probes

The term “probe” designates a substance that can specifically binds to atarget, wherein the target may be a biological marker, another probe,reporter, or any molecule associated with said biological marker,another probe or reporter.

In one embodiment the probe is an antibody probe.

Antibody, as used herein, means an immunoglobulin or a part thereof, andencompasses any polypeptide comprising an antigen-binding siteregardless of the source, method of production, and othercharacteristics. The term includes for example, polyclonal, monoclonal,monospecific, polyspecific, humanized, single-chain, chimeric,synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies. Apart of an antibody can include any fragment which can still bindantigen, for example, an Fab, F(ab′)₂, Fv, scFv. The origin of theantibody is defined by the genomic sequence irrespective of the methodof production.

Primary antibody, as used herein, refers to an antibody thatspecifically binds to a biological marker of interest present within thebiological sample. In certain embodiments the primary antibody may bepolymerized. Primary antibodies may be derived from any warm bloodedspecies, e.g. mammals, birds.

Secondary antibody, as used herein, refers to an antibody that has anantigen binding domain that specifically binds to a first probe, e.g., aprimary antibody, or a hapten deposited in the target site, or haptenlinked directly or indirectly to a first probe.

Tertiary antibody, as used herein, refers to an antibody that has anantigen binding domain that specifically binds to a second probe (e.g.,a secondary antibody) or a hapten linked to a second probe or a haptenlinked to polymer conjugated to a second probe, or hapten deposited inthe target site.

Sometimes an antibody may function both as a secondary and a tertiaryantibody.

Antibodies used in the invention, including primary antibodies,secondary antibodies and tertiary antibodies, may be derived from anymammal species, e.g., a rat, a mouse, a goat, a guinea pig, a donkey, arabbit, horse, lama, camel, or any avian species e.g., chicken, duck.Derived from any mammal or avian species, as used herein, means that atleast a part of the nucleic acid sequence encoding a particular antibodyoriginated from the genomic sequence of a specific mammal, e.g., a rat,a mouse, a goat, or a rabbit or a specific bird e.g., chicken, duck. Theantibody may be of any isotype, e.g., IgG, IgM, IgA, IgD, IgE or anysubclass, e.g., IgG1, IgG2, IgG3, IgG4.

In certain embodiments the primary antibody contains an antigen bindingregion which can specifically bind to a biological marker expressed bycells comprising a biological sample. The marker may be expressed on thecell surface or within the cell membrane, i.e., on the interior of thecell, e.g., within the cytoplasm, within the nucleus, within theendoplasmic reticulum. In some embodiments the biological marker issecreted from the cell and thus is present in solution, e.g., in cellculture media, in blood or plasma.

In certain embodiments, the secondary antibody contains an antigenbinding region which specifically binds to the primary antibody, e.g.,the constant region of the primary antibody.

In certain embodiments, the secondary antibody is conjugated to apolymer. In some embodiments, the polymer is conjugated with 2-20secondary antibodies. In other embodiments, the polymer is conjugatedwith 2-10 secondary antibodies.

In certain embodiments, the tertiary antibody contains an antigenbinding region which specifically binds to the secondary antibody, e.g.,a constant region of the secondary antibody, or a hapten linked to thesecondary antibody or a polymer conjugated to the secondary antibody. Incertain embodiments, the tertiary antibody is conjugated to a polymer.In some embodiments, the polymer is conjugated with 1-20 tertiaryantibodies. In other embodiments, the polymer is conjugated with 1-5tertiary antibodies.

The antibodies that may be used in the methods and compositions of theinvention include monoclonal and polyclonal antibodies, engineeredantibodies including chimeric, CDR-grafted and artificially selectedantibodies produced using phage display or alternative techniques.

Various techniques for producing antibodies have been described, see,e.g., Kohler and Milstein, (1975) Nature 256:495; Harlow and Lane,Antibodies: a Laboratory Manual, (1988) (Cold Spring Harbor Press, ColdSpring Harbor, N.Y.) incorporated herein by reference. Techniques forthe preparation of recombinant antibody molecules is described in theabove references and also in, for example, EP 0623679; EP 0368684; andEP 0436597.

Antibodies may be produced recombinantly or synthetically. Nucleic acidsencoding antibodies may be isolated from a cDNA library. Nucleic acidsencoding antibodies may be isolated from a phage library (see e.g.McCafferty et al. 1990, Nature 348:552, Kang et al. 1991, Proc. Natl.Acad. Sci. USA 88:4363; EP 0 589 877 B1). Nucleic acids encodingantibodies can be obtained by gene shuffling of known sequences (Mark etal. 1992, Bio/Technol. 10:779). Nucleic acids encoding antibodies can beisolated by in vivo recombination (Waterhouse et al. 1993, Mid. AcidRes. 21:2265). The antibodies used in the methods and compositions ofthe invention include humanized immunoglobulins (U.S. Pat. No.5,585,089, Jones et al. 1986, Nature 332:323).

The antibodies may be altered antibodies comprising an effector proteinsuch as a toxin or a label, e.g., a detectable substance.

Antibodies may be obtained from animal serum, or, in the case ofmonoclonal antibodies or fragments thereof, produced in cell culture.Recombinant DNA technology may be used to produce the antibodiesaccording to established procedure, in bacterial, yeast, insect ormammalian cell culture. In certain embodiments, the selected cellculture system preferably secretes the antibody product.

Nucleic Acid Probes

In another embodiment a first probe may be or comprise a nucleic acid ornucleic acid analog molecule, e.g., a DNA molecule, an RNA molecule, aPNA molecule, for use in in situ hybridization. Nucleic acid probes maybe synthesized chemically or produced recombinantly in cells (see e.g.Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.Cold Spring Harbor Press). In some embodiments, the probe is comprisedof a peptide nucleic acid (PNA). A peptide nucleic acid is a nucleicacid molecule in which the deoxyribose or ribose sugar backbone, usuallypresent in DNA and RNA is replaced with a peptide backbone. Methods ofmaking PNAs are known in the art (see e.g. Nielson, 2001, CurrentOpinion in Biotechnology 12:16) (hereby incorporated by reference). Inother embodiments, the probe is comprised of locked nucleic acids (LNA)(Sorenson et al. 2003, Chem. Commun. 7(17):2130). In some embodiments,the nucleic acid probe specifically binds to the biological marker,e.g., a nucleic acid molecule contained within the biological sample.

The nucleic acid probe, in particular embodiments, comprises at least asequence that specifically hybridizes to a target sequence in thebiological sample, e.g. a nucleic acid sequence such as a genomic DNAsequence or an mRNA sequence, under specific conditions of stringency.As used herein, the term “hybridization under stringent conditions,” isintended to describe conditions for hybridization and washes under whichnucleotide sequences that are significantly complementary to each otherremain bound to each other. The conditions are such that sequences atleast 70%, at least 80%, at least 85-90% complementary remain bound toeach other. The percent complementary is determined as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389-3402 (herebyincorporated by reference).

Specified conditions of stringency are known in the art and can be foundin Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(Ausubel et al. 1995 eds.), sections 2, 4, and 6 (hereby incorporated byreference). Additionally, specified stringent conditions are describedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nded. Cold Spring Harbor Press, chapters 7, 9, and 11 (hereby incorporatedby reference). In some embodiments, the hybridization conditions arehigh stringency conditions, An example of high stringency hybridizationconditions is hybridization in 4×sodium chloride/sodium citrate (SSC) at65-70° C. or hybridization in 4×SSC plus 50% formamide at 42-50° C.,followed by one or more washes in 1×SSC, at 65-70° C. It will beunderstood that additional reagents may be added to hybridization and/orwash buffers, e.g., blocking agents (BSA or salmon sperm DNA),detergents (SDS), chelating agents (EDTA), Ficoll, PVP, etc.

In some embodiments, the nucleic acid probes hybridize to a targetsequence in a sample under moderately stringent conditions. Moderatestringency, as used herein, include conditions that can be readilydetermined by those having ordinary skill in the art based on forexample, the length of the DNA. Exemplified conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press (1989) (herebyincorporated by reference), and include use of a prewashing solution of5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of 50%formamide, 6×SSC at 42° C. (or other similar hybridization solution,such as Stark's solution, in 50% formamide at 42° C.), and washingconditions of 60° C., 0.5×SSC, 0.1% SDS.

In some embodiments, the nucleic acid probes hybridize to a targetsequence in a sample under low stringent conditions. Low stringencyconditions may include, as used herein, conditions that can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. Low stringency may include, for example,pretreating the DNA for 6 hours at 40° C. in a solution containing 35%formamide, 5 x SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1%Ficoll, 1% BSA, and 500 ug/ml denatured salmon sperm DNA. Hybridizationsare carried out in the same solution with the following modifications:0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 pg/ml salmon sperm DNA, 10%(wt/vol) dextran sulfate, and 5-20×10⁶ CPM probe is used. Samples areincubated in hybridization mixture for 18-20 hours at 40° C., and thenwashed for 1.5 h at 55° C. in a solution containing 2 x SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.

Other embodiments of the probes include peptide sequences, e.g. peptidesequences derived from protein or nucleic acid binding domains ofdifferent proteins, ligands of different cellular and nuclear receptorsand their derivatives, small molecules which can bind specifically tocertain structural units of large biological molecules, however, this isjust a list of non-limiting examples of substances that can be used asprobes for the purposes of the present invention.

EXAMPLES 1. Examples of Reporter and Cross-Linker Molecules of theInvention. ABBREVIATIONS

MBHA 4-Methylbenzhydrylamine

NMP N-Methyl Pyrolidon

HATU 2-(1h-7-azabenzotriazole-1-yl)-1,1,3,3 tetramethyluroniumhexafluorophosphate; rnethenamminium

DIPEA Di/sopropyl EthylAmine

DCM Dichloro Methane

TFA TriFluoroacetic Acid

TFMSA TriFluor Methyl Sulphonic Acid

Fer Ferrulic acid

FLU Fluorescein

Tyr Tyrosine

Lys Lysine

Dex Dextran

HPLC High Performance Liquid Chromatography

equi. equivalent

1.1. Fer-L30-Lys(Flu)-NH₂ (D17158)

MBHA resin was downloaded with Fmoc-Lys(ivDDE) to a loading of 150 micromol/g. 200 mg resin was de-Fmoc'ed with 20% piperidine in NMP, thesubjected to one coupling with Boc-L30-OH (1.5 mL 0.26 M in NMP,preactivated with 0.9 equi. HATU, 2 equivalents DIPEA for 2 min) for 20min. The ivDDE group was removed with 5% hydrazine in NMP, and thelysine side chain was labeled with carboxy fluorescein (Flu) (1.5 mL 0.2M in NMP, preactivated for 2 min with 0.9 equi. HATU, 2 equi DIPEA) for2×20 min. The resin was treated with 20% piperidine in NMP, NMP, DCMthen DCM. The intermediate product H-L30-Lys(Flu)-NH₂ was cleaved of theresin with TFA:TFMSA:mCresol (7:2:1, 1.5 ml for 1 h), precipitated withdiethyl ether, re-suspended in TFA, precipitated with diethyl ether,re-suspended in NMP and again precipitated with diethyl ether. It wasmade basic with 100 microL DIPEA and dissolved directly in 0.5 mL 0.3MFerulic acid preactivated with 0.9 equi. HATU and 2 equi. DIPEA. After25 min the crude product was precipitated with diethyl ether, dissolvedin 450 microL NMP and 50 microL ethylenediamine. After 5 min the productwas precipitated with diethyl ether, dissolved in 15% acetonitril inwater (8mL) and acidified with 100 microL TFA and subjected to RP-HPLCpurification.

1.2. Fer-L150-Lys(Flu)-NH₂ (D17157)

MBHA resin was downloaded with Boc-Lys(Fmoc) to a loading of 100 micromol/g. 100 mg resin was subjected to 5 coupling cycles with Boc-L30-OH(a. Coupling with Boc-L30-OH as in 1. b. Capping with 2% aceticanhydride in NMP: Pyridine 1:1, 2 min. c. De-Bc with 5% mCresole in TFA2×5 Min.) . The lysine side chain was De-Fmoc'ed and labeled withcarboxy fluoresceine, as in 1. The intermediate productH-L150-Lys(Flu)-NH₂ was cleaved of the resin, and labeled N-terminallywith Ferulic Acid and purified as in 1.1.

1.3. betaala-L90-Lys(Flu)-L9OLys(Flu)-L90-Lys(Flu)-NH2 (D16127)

Boc-L90-Lys(Fmoc)-L90-Lys(Fmoc)-L9OLys(Fmoc) was prepared on 0.5 g MBHAresin with standard solid phase chemistry (as in 1.1. and 1.2). Fmocgroups were removed from lysine side chains with 20% piperidine in NMPand the compound was subjected to repeated carboxy fluorescein labeling(3×30 min). Following removal Boc groups with TFA, the N-terminal waslabeled on solid phase with betaalanine-N,N-di acetic acid (betaala)tert-butyl ester. Following cleavage from resin and HPLC purification,betaala-L90-Lys(Flu)-L9OLys(Flu)-L90-Lys(Flu)-NH2 was isolated.

1. 4. H-Cys-L90-Lys(Flu)-L9OLys(Flu)-L90-Lys(Flu)-NH2 (D16126)

Boc-L90-Lys(Fmoc)-L90-Lys(Fmoc)-L9OLys(Fmoc) resin was prepared andlabeled with fluorescein using the procedure described in 1.3. Followingremoval Boc groups the N-terminal was labeled withN-Boc-S(4-Methoxybenzyl)-Cys-OH. The compound was cleaved from thecolumn and purified by HPLC:

Molecules 1.1, 1.2, 1.3. and 1.4. are non-limited embodiments ofreporters comprising Fluorescein residues.

1.5.Fer-Lys(Fer)-L30-Lys(Fer)-L30-Lys(Fer)-L30-Lys(Fer)-L30-Lys(Fer)-L3OLys(NH2)-NH2(D17120)

To MBHA resin was sequentially coupled Boc-Lys(Fmoc) (2 cycles),Boc-L30-OH (5 cycles) and Boc-Lys(2CIZ)-OH. The intermediate product wascleaved from the resin in the presence of 10% thioanisol scavenger toremove 2CIZ-groups. The N-terminal and the 5 de-protected lysine sidechains were labeled with Ferulic acid as in 1.1 (2×30 Min). The Fmocgroup on the N of the C-terminal Lysine residues was then removed with10% ethylene diamine in NMP prior to purification.

1.6. Fer-(Lys(Fer)-L30)₅-Lys(NH-betala((L90-Lys(Flu))3-NH₂)-NH₂(D17134)

betaala-L90-Lys(Flu)-L9OLys(Flu)-L90-Lys(Flu)-NH2 (compound 1.4) 500nmol was dissolved in 88 microL NMP and 2 microL pyridine, and convertedto cyclic anhydride by reaction with 10 microL diisopropyl carbodiimiidefor 10 min. The anhydride was precipitated with diethyl ether, and thepellet was dissolved in 100 microL NMP comprising 250 nmolFer-(Fer-L30)₅-Lys(NH₂)-NH₂. After 20 min 5 microL ethylene diamine wasadded, and after 5min the product was precipitated with diethyl ether,acidified and HPLC purified.

1.7. Ac-(Tyr(OH)-L30)₅-L90-Lys(Flu)-L90-Lys(Flu)-Lys(Flu)-NH₂ (D18044)

Ac-(Tyr(2BrZ)-L30)₆-L90-Lys(Fmoc)-L90-Lys(Fmoc)-Lys(Fmoc) was preparedon MBHA resin. On solid phase the Fmoc groups were removed, and thelysine side chains labeled with carboxy fluorescein. Following cleavagefrom the resin, the product was HPLC purified.

1.8.Fer-Lys(Fer)-L60-Lys(Fer)-Lys(Fer)-L60-Lys(Fer)-Lys(Fer)-L3OLys(NH₂)-NH₂ (D17140)

Boc-Lys(2CIZ)-L60-Lys(2CIZ)-Lys(2CIZ)-L60-Lys(2CIZ)-Lys(2CIZ)-L30-Lys(Fmoc)was prepared on MBHA resin. Following cleavage from the resin, theintermediate productH-Lys(NH₂)-L60-Lys(NH₂)-Lys(NH₂)-L60-Lys(NH₂)-Lys(NH₂)-L30-Lys(Fmoc) wasisolated by precipitation, and labeled with Ferulic acid as in 1.1. Thefinal product was isolated by HPLC.

Examples 1.5.-1.8. are the examples of cross-linker molecules of theformula

(R1)n-(X)q-R2(m),

-   -   wherein    -   R1 and R2 are different moieties of HRP substrate(s) (e.g. Fer        or Tyr)    -   X is a linker molecule of the following formula

-   -   wherein R3 and R4 are residues of Lys,

and m, n and q are from 1 to 6.

Linear polymer cross-inkers (for example molecules 1.6. and 1.7,described above) bearing several residues of fluorescein, Ferulic acid(1.6) and/or tyrosine (1.7) are also embodiments of reporters that canbe deposited by HRP in the presence of another cross-linker, e.g. DAB.Such linear relatively low molecular weight (MW<15 kDa)cross-linkers/reporters may be particular useful when the HRP enzymemoieties are bound to target sites which are not easily accessible orexposed, e.g. in cell nuclei such as DNA. Larger reporter molecules, forexamples reporters comprising dextran molecules conjugated with tens andhundreds of labels (e.g. Flu and/or Fer and/or Tyr), such as for examplemolecule 1.9. (described below), may be used when peroxidase enzymemoieties are located in more easy accessible target sites.

1.9. Dex70 Conjugated with Cross-Linker 1.5. and Cross-Linker 1.4.(D17130)

Dextran MW 70kDa, activated with divinyl sulphone, 10 nmol, was reactedwith Fer-(Fer-L30)₅-Lys(NH₂)-NH₂ (compound 1.5) 500 nmol, in a totalvolume of 300 microL 0.16M NaHCO₃ pH 9.5 for 30 min at 40 C. After aslight precipitation was observed, further 100 microL water was addedand the reaction was allowed to proceed for another 30 min. Further 200microL 0.15 M NaHCO₃ was added together with 500 nmolH-Cys-L90-Lys(Flu)-L9OLys(Flu)-L90-Lys(Flu)-NH2 (compound 1.4). After 1h at 40 C, the reaction mixture was quenched by addition of 50 microL0.165M cystein for 30 min, solution was filtered, and the product waspurified by FPLC on superdex 200 with 20% EtOH in aqueous solutioncontaining 10 mM CHES, pH 9.0, and 0.1 M NaCl. The product eluted was aDextran conjugate comprising around 56 Fluorescein and 113 Ferulic Acidresidues.

1.10. Goat-Anti-Mouse-Dex70-HRP (D18033)

13.7 nmol divinylsulphone were activated 70 kDA MW dextran and reactedwith 602 nmol 5 HRP were in 600 microL buffer (100 mM NaCl, 25 mMNaHCO₃, pH 9.5) for 3 h at 30 C. Then 41.1 nmol Goat-anti-Mouse F(ab)₂antibody in 105 microL water was added, and the reaction was continuedfor additional 16 h. The reaction mixture was quenched by addition of 70microL 0.165M cystein for 30 min and the product was purified onsuperdex 200 in 100 mM NaCl, 10 mM HEPES pH 7.2. The eluded product wasa Dextran conjugate comprising Goat-anti-Mouse (GaM) and HRP (Ratiodex:GaM:HRP=1:1:1).

1.11. Anti-FITC-Dex70-HRP (D18058)

10 nmol divinylsulphone activated 70 kDA MW dextran and 440 nmol HRPwere reacted in 400 microL buffer (100 mM NaCl, 25 mM NaHCO₃, pH 9.5)for 3 h at 30 C. Then, 30 nmol Anti-Mouse F(ab)₂ antibody in 80 microLwater was added, and the reaction was continued for additional 90 min at40 C. The reaction mixture was quenched by addition of 50 microL 0.165Mcystein for 30 min and the product was purified on superdex 200 in 100mM NaCl, 10 mM HEPES pH 7.2. The eluded product was a conjugate ofDextran with anti-FITC and HRP (Dex/anti-FITC/HRP Ratio=1/2/9).

1.12. Anti-FITC-Dex70-HRP (D17030)

10 nmol divinylsulphone activated 70 kDA MW dextran ;440 nmol HRP and 25nmol F(ab)₂ anti-FITC were reacted in 374 microL buffer (100 mM NaCl, 25mM NaHCO₃, pH 9.5) for 16 at 30 C. The reaction mixture was quenched byaddition of 50 microL 0.165M cystein for 30 min and the product purifiedon superdex 200 in 100 mM NaCl, 10 mM HEPES pH 7.2. The eluded productwas a Dextran conjugate comprising Anti-FITC and HRP (ratio 1:1:1).

Molecules 1.10, 1.11 and 1.12 represent embodiments ofantibody-Dextran-HRP conjugates which may be useful as probes fordetection of deposited reporter molecules. Conjugate 1.10. may also beuseful as a further probe for the detection of the probes bound to thedeposited reporter (e,g. on step (b) of the method for detecting amarker in the target site described above).

3. Example of IHC Stainings Using the Method of the Invention.

IHC was carried out on formalin fixed paraffin embedded tonsils. 3-5micron sections were cut, baked and stored at 4 C, until used. Paraffinwas then removed by xylene (2×5 min); 99% ethanol (2×2 min); 70% ethanol(2×2 min) and finally water. The slides were put in the target retrievalsolution, pH 9, (DAKO S2367) then heated in microwave oven (boiled for10 min). Afterwards, the slides were allowed to cool and then weretransferred to the wash buffer (DAKO S3006). The procedure was followedby a step of blocking of endogenous Peroxides activity with 3% hydrogenperoxide for 5 min., then again the slides were transferred into thewash buffer and then stained. To minimize slide to slide variation eachcomparative experiment was carried out with consecutively cut sectionsduring the same day. The specific as well as background signals werescored using a score scale from 0 to 4 wherein 0 is representing nostain at all, 1—weakly stained, 2—moderately stained, 3—stronglystained, 4—over stained.

IHC Experiment 1.

Cytokeratin, Goat-anti-Mouse-HRP and Anti-FITC-HRP each were diluted in2% BSA, 0.2% Casein, 2% PEG, 0.1% Tween20, 0.1 M NaCL, 10 mM HEPES, pH7.2. (BCPT-buffer). Reporter D17128 and DAB (DAKO K5007 C) were dilutedin DAB substrate buffer (DAKO K5007 B). All incubations lasted 5 min,followed by 2 min wash in DAKO washing buffer S3006, except for afterthe incubation with 017128, where washes were performed in 10 mM CHES pH9+0.1% Tween20.

Table and text below summarize the embodiments of the experiment:

Primary Secondary Reporter Reporter antibody antibody Cross-linkerdetection Cross-linker detection Developing probe probe and reporterprobe and reporter probe solution Slide 1 Cytokeratin GeM- HRP, DAB(DAKO D18033, (DAKO M5315) 15 125 nM, K5007 C) nM No reporter Slide 4 Asslide 1 GaM-HRP, D17128 D17030 DAB D18033 5 100 nM + 50 nM nM DAB 1:150Slide 7 As slide 1 GaM-HRP, As slide 4 As slide 4 D 17128 D 17030 DABD18033 100 nM + 50 nM 0.5 nM DAB 1:150 Slide 9 As slide 1 GaM-HRP,D17128 As slide 4 As slide 7 As slide 7 □AB D18033 20 nM + 0.5 nM DAB1:150 Slide 12 As slide 9 D17128 As slide 9 DAB 25 nM + (DAKO D17140K5007 C) 100 microM Slide 14 As slide 9 D17128 As slide 9 DAB 25 nM +(DAKO DAB 1:150 K5007 C)

Slide 1—standard DAB-HRP staining (no amplification), Standard DAB DAKOreagent (K7005 C) contains 3mM DAB.

Slide 4—deposition of reporter D17128 in the presence of 1mM DAB-GaM-HRPconjugate D18033 diluted 25 times compared to slide 1—one stepamplification Slide 7 and Slide 9—further dilution of GaM-HRP conjugateD18033 compared to slide 1 and additional step of reporter deposition.

Slides 1, 4 and 7 were all specifically stained scoring from 2.5 to 3. A250 times amplification of the signal (slide 9 compared to slide 1) wasachieved due to deposition of reporter D17128 in the presence of DABfollowed by recognition the reporter by Anti-FITC-HRP probe D17030.Remarkably the stains of both slides 7 and 9 remained crisp (i.e.non-diffused), with sharp borders between stained and unstained areasindicating that very little diffusion takes place in the depositionsites.

IHC Experiment 2.

Cytokeratin, GaM-HRP and Anti-FITC-HRP were all diluted in 2% BSA, 0.2%Casein, 2% PEG, 0.1% Tween20, 0.1 M NaCL, 10 mM HEPES, pH 7.2.(BCPT-buffer). D17128 and DAB (DAKO K5007 C) were diluted in DABsubstrate buffer (DAKO K5007 B). All incubations were 5 min, followed by2 min wash in DAKO S3006, except following the incubation with reporterD17128, wherein the washes were performed in 10 mM CHES pH 9+0.1%Tween20.

The table and text below summarize the embodiments of the experiment:

Primary & secondary Cross-linker & Reporter Developing antibody probesreporter probe solution Slide 9 Cytokeratin D17128 25 nM no D18058Anti-FITC-HRP DAB DAKO M5315 cross-linker 25 nM (DAKO 15 nM + GaM-HRPK5007 C) D18033 10 nM Slide 12 As slide 9 D17128 25 nM + As slide 9 DABD17140 (DAKO 100 microM K5007 C) Slide 14 As slide 9 D17128 25 nM + Asslide 9 DAB DAB 1:150 (DAKO K5007 C)

Slides 12 and 14 were strongly and specifically stained (both scoring2), whereas slide 9 was not stained (score 0). The results show that thereporter is not precipitated by HRP in the media without a cross-linker(e.g. DAB (slide 14) or D17140 (slide 12)).

The experiment further illustrates the possibility of reducing thenumber of steps by pre-incubating (for 10 min) primary antibody withsecondary antibody-HRP conjugate.

IHC Experiment 3.

Cytokeratin, GaM-HRP and Anti-FITC-HRP each were diluted in 2% BSA, 0.2%Casein, 2% PEG, 0.1% Tween20, 0.1 M NaCL, 10 mM HEPES, pH 7.2.(BCPT-buffer). D17128 and DAB (DAKO K5007 C) were diluted in DABsubstrate buffer (DAKO K5007 B). All incubations were 5 min, followed by2 min wash in DAKO S3006, except following the incubation with D17128and D18044, wherein washes were performed in 10 mM CHES pH 9+0.1%Tween20. When the third step of incubation of the slides with DAB insubstrate buffer was applied (slides 3 and 5), the incubation time was 1min.

The table and text below summarize the embodiments of the experiment:

Primary Secondary antibody antibody Cross-linker Cross-linker ReporterDeveloping probe probes and reporter and reporter probe solution Slide 2Cytokeratin GAM-HRP, D17128 D18058 DAB DAKO D18033 50 nM + Anti- M5315 3nM DAB FITC- 15 nM 1:150 HRP 50 nM Slide 3 As slide 2 As slide 2 DABD17128 D18058 DAB No 50 nM A- Anti- reporter DAB FITC-HRP 1:150 50 nMSlide 4 As slide 2 As slide 2 D17128 D18058 DAB 50 nM No Anti- cross- FITC-HRP linker 50 nM Slide 5 As slide 2 As slide 2 DAB D17128 D18058 DABNo 50 nM Anti- reporter No cross- FITC-HRP linker 50 nM

Slide 2 was the most intensely stained slide (score 3), showing that theone minute of extra application of DAB of the third step on slide 3(score 2,5) did not much improve, but rather slightly reduced thestaining intensity. The lack of staining in absence of cross-linker onslide 4 (score 0) is in accordance with the results obtained in IHCexperiment 2 (described above). On slide 5, where DAB was applied priorto application of the reporter conjugate D17128, a staining was observed(score 2), albeit not quite as intense as on slide 3.

This results show that a cross-linker can be first applied in a separatestep before incubating the slide with both reporter and cross-linker(i.e. on pre-incubation step). Another positive effect of applying DABprior to depositing the labeled reporter observed was a remarkablereduction of the background staining (scored 0.5-1 on slide 2, andscored 0 on slide 3), while specific signals intensity did not decreasemuch, Particularly weak diffuse background staining associated withunspecific binding of Goat-anti-Mouse- HRP conjugates, was in many casesvirtually eliminated by the 1 min extra DAB step prior to deposition.

IHC Experiment 4.

Cytokeratin, GaM-HRP and Anti-FITC-HRP were all diluted in 2% BSA, 0.2%Casein, 2% PEG, 0.1% Tween20, 0.1 M NaCL, 10 mM HEPES, pH 7.2.(BCPT-buffer). D17128, D18044 and DAB (DAKO K5007 C) were diluted in DABsubstrate buffer (DAKO K5007 B). All incubations were 5 min, followed by2 min wash in DAKO S3006, except following incubations with D17128 andD18044, wherein washes were performed in 10 mM CHES pH 9 +0.1% Tween20.

The table and text below summarize the embodiments of the experiment:

Primary Cross- &secondary Preincubation linker antibody with cross-(DAB) & probes linker (DAB) reporter Slide 8 Cytokeratin DAB 1:50 inD17128 50 nM DAB DAKO M5315 substrate 50 nM + D 18058 15 nM + GaM-buffer (DAKO DAB 1:50 HRP D18033 K5007 C), (DAKO 10 nM 1 min. K5007 C)Slide 9 As slide 8 As slide 8 D17128 50 As slide 8 As slide 8 nM + DAB1:150 Slide 10 As slide 8 As slide 8 D17128 50 As slide 8 As slide 8nM + DAB 1:500 Slide 11 As slide 8 As slide 8 D 18044 5 As slide 8 Asslide 8 microM + DAB 1:50 Slide 12 As slide 8 As slide 8 D18044 5 Asslide 8 As slide 8 microM + DAB 1:150 Slide 13 As slide 8 As slide 8D18044 5 As slide 8 As slide 8 microM + DAB 1:500

This experiment illustrates the effect of cross linker concentration ondeposition of two different types of reporters. Slide 9 was the mostintensely stained slide (score 4). DAB as cross linker was applied onslide 9 at dilution 1:150, (1 mM). A higher (1:50 (3mM), slide 8) orlower (1:500 (0.3mM), slide 10) concentrations of DAB gave slightly lessintense staining (score 3.5). The background reducing effect of thepre-incubation with DAB was also visible; none of the six slides (of theabove table) had significant background staining.

The low molecular weight reporters (e.g. reporter 18044 described above)were better deposited at lower concentrations of DAB (slide 13; score3); the staining intensity decreased with increasing DAB concentrations(slide 12, score 2.5; slide 11, score 2)

This experiment also illustrates that larger reporter moleculescomprising many peroxidase substrate moieties can be used at loweramounts (e.g. Fluorescein-Ferulic Acid Dextran conjugate D17128, 50 nM)compared to smaller reporter molecules comprising the same but fewerperoxidase substrate moieties (e.g. D 18044,t 5 microM) to achieve thesame or even better staining results.

4. Example of Amplification of a Nucleic Acid Probe Signal Using theMethod of the Invention.

By running samples with and without the use of DAB in the Chromogensolution and the signal enhancer solution the precipitating effect ofDAB can be illustrated. Further illustration of the inventions signalamplification can be obtained by further dilution of either the probe,the antibody or by omitting the second Peroxidase-blocking step.

Tissue: Formalin-fixed Paraffin embedded Tonsil. Fluorescein labeledprobe targeted against the centromer of Chromosome 11. The followingsolutions are used in the example:

Antigen retrieval/pre-treatment (Dako K 5599) Washbuffer1 (Dako K 5331)Washbuffer2 (Dako S 3006) Pepsin RTU (Dako K 5331) Stringent washbuffer,(Dako K 5331) R-a-Fitc/HRP, F(ab) (Dako P 5100) Antibody diluent (Dako S2022) Probe (Dako Y 5505) Peroxidase-Blocking solution (Dako S 2023)Nuclear Fast red (Dako S1968) Chromogen buffer (Dako K5007)

A Hybridizer(Dako S2451) was used for hybridising the probes

Preparation of the Chromogen Solution and the Signal Enhancer Solution:Solution A:

102.5 mg5-amino-2[3-[5-amino-1,3-dihydro-3,3-dimethyl-1-(4sulfobutyl)-2Hindol-2-ylidene]-1-propenyl]-3,3dimethyl-1-(4sulfobutyl)-3H-Indolium,tertrifluoroacetate is dissolved in 4,525 mL Propanediol/water 8:2

Solution B:

59.6 mg 3,3′-Di-aminoberizidine dihydrochloride is dissolved 5.96 mLPropanediol/water 8:2 13.7 μL. 12 M Hydrochloric acid is added.

Solution C:

Solution A and Solution B are mixed in the ratio 1 mL A: 9 mL B.

Chromogen Solution:

For staining these stock solutions were diluted into the chromogenbuffer of kits obtained from Dakocytomation code #K5007.

1 mL Solution C is mixed with 19 mL Chromogen buffer

Solution D:

1 mL Solution B is diluted with 499 mL Chromogen buffer.

Signal Enhancer Solution:

This is a solution of D1734 in 20% ethanol, 0.1 M NaCl, 10 mM CHES pH9.0 diluted with Solution D to a concentration of 250 nM or 50 nM ofFer-L30-FITC. In the case of No DAB in the solution dilution wasperformed directly in Chromogen buffer

Procedure for Tissue Staining:

Human Tonsil tissue slides were de-paraffinized, washed and antigenretrieval was performed in 10 min in a microwave oven. After anotherwash, pepsin treatment at 37° C. and washing, slides where dehydratedand incubated with the PNA probe. After 5 min denaturing of the sampleat 85° C., the probes where hybridized for 1 hour at 45° C. The slideswere stringent washed at 65° C. for 10 min. Peroxidase was blocked for 3min, the slides were washed and incubated with an Rabbit-anti-FITC/HRPdiluted 1:20. After 30 min the slides where washed and incubated withthe signal enhancer solution. After 30 min the slides where washed,peroxidase was blocked for 3 min, washed again and the slides wereincubated with an Rabbit-anti-FITC/HRP diluted 1/20. After 30 min theslides were washed and incubated with a Chromogen solution. Followingwashing with water the slides were counterstained with nuclear fast red,washed and mounted.

The experimental set-up and results of the staining are illustrated inthe following table:

anti- Signal anti- FITC/HRP enhancer FITC/HRP Result Slide ProbeAntiboby solution Antiboby Signal- #. 1 time 30 min 30 min 30 minintensity Comments 1 Buffer Anti Fer₆-Flu₃ 1:20 0 FITC/HRP 50 nM 1:50 with DAB 2 Y5505 1:100 Fer₆-Flu₃ 1:20 0 Diluted 10 X 50 nM 3 Y5505 1:100Fer₆-Flu₃ 1:20 1½ No DAB in the Fortyndet 50 nM chromogen 50 x with DABsolution 4 Y5505 1:100 Fer₆-Flu₃ 1:20 2½ Diluted 10 x 50 nM with DAB 5Y5505 1:100 Fer₆-Flu₃ 1:20 4 Over stained/ Diluted 10 x 250 nM to muchsignal with DAB 6 Y5505 1:100 Fer₆-Flu₃ 1:20 0½ Diluted 10 x 250 nM 7Y5505 1:100 No amplification stained 0 Fortyndet directly after first 10x incubation with anti FITC/HRP

Slide 1 is a control showing probe is need to get a signal

Slide 2 shows that without a cross-linker in the signal enhancingsolution no specific signal is obtained

Slide 3 shows that adding a cross-linker (DAB) to the signal enhancingsolution results in high signal amplification. This is the only slidethat did not have DAB in the chromogen solution.

Slide 4 shows that adding a cross-linker to the chromogen solutionfurther enhances the signal (compared to slide 3)

Slide 6 shows that by increasing the concentration of the signalenhancer (D17134) a weak signal can be obtained without a crosslinker.However adding a cross-linker gives a strong amplification of thissignal (as illustrated by slide 5)

1-49. (canceled)
 50. A method of detecting of a biological marker in abiological sample in vitro, comprising the following steps: a)incubating a biological sample presumably comprising a biological markerwith one or more probes capable of specifically binding to thebiological marker, wherein -least one of said one or more probescomprises at least one moiety of horse radish peroxidase (HRP), therebyforming a complex of the biological marker with the at least one probecomprising at least one moiety of HRP; b) incubating the samplecomprising the complex of (a) in a water solution, comprising3,3′-diaminobenzidine, a peroxide compound and a conjugate moleculecomprising comprising a combination of different detectable labels,wherein the combination comprises a hapten and a substrate of horseradish peroxidase, thereby depositing said conjugate molecule in a siteof the sample where said complex is present; c) detecting the depositedconjugate molecules of (b) and thereby detecting the biological marker.51. The method according to claim 50, wherein the biological marker is abiological molecule selected from the group consisting of proteins,nucleic acids, lipids, carbohydrates, or is a molecular complex or apolymer comprising two or more said biological molecules or derivativesthereof, or is a cellular structure.
 52. The method according to claim50, wherein the probe is a member of a specific binding pair or is aconjugate comprising a member of a specific binding pair, wherein saidmember of a specific binding pair is capable of specifically binding tothe biological marker.
 53. The method according to claim 50, wherein anamount of 3,3′-diaminobenzidine is from about 0.3 mM M to about 3 mM.54. The method according to claim 50, wherein the method comprises anadditional step between step (a) and step (b), wherein the sample isincubated in a water solution comprising 3,3′-diaminobenzidine in anamount from 0.3 mM to 3 mM and a peroxide compound
 55. The methodaccording to claim 50, wherein step (c) comprises incubating the samplewith a substance which is capable of specifically binding to thedeposited conjugate.
 56. The method according to claim 55, wherein thesubstance is detectably labeled.
 57. The method according to claim 56,wherein the substance is an antibody or a nucleic acid.
 58. The methodaccording to claim 57, wherein the method is performed in a format of animmunohistochemistry or in situ hybridization assay.